Ultra-sensitive detection of molecules or particles using beads or other capture objects

ABSTRACT

The present invention relates to systems and methods for detecting analyte molecules or particles in a fluid sample and in some cases, determining a measure of the concentration of the molecules or particles in the fluid sample. Methods of the present invention may comprise immobilizing a plurality of analyte molecules or particles with respect to a plurality of capture objects. At least a portion of the plurality of capture objects may be spatially separated into a plurality of locations. A measure of the concentration of analyte molecules in a fluid sample may be determined, at least in part, on the number of reaction vessels comprising an analyte molecule immobilized with respect to a capture object. In some cases, the assay may additionally comprise steps including binding ligands, precursor labeling agents, and/or enzymatic components.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/265,996, filed Sep. 15, 2016, entitled “Ultra-Sensitive Detection ofMolecules or Particles Using Beads or Other Capture Objects,” by Duffyet al., now abandoned, which is a continuation of U.S. patentapplication Ser. No. 13/530,979, filed Jun. 22, 2012, now U.S. Pat. No.9,482,662, entitled “Ultra-Sensitive Detection of Molecules or ParticlesUsing Beads or Other Capture Objects,” by Duffy et al., which is acontinuation of U.S. patent application Ser. No. 12/731,130, filed Mar.24, 2010, now U.S. Pat. No. 8,236,574, entitled “Ultra-SensitiveDetection of Molecules or Particles Using Beads or Other CaptureObjects,” by Duffy et al., which claims the benefit of U.S. ProvisionalPatent Application Ser. No. 61/309,141, filed Mar. 1, 2010, entitled“Ultra-Sensitive Detection of Molecules or Particles Using Beads orOther Capture Objects,” by Duffy et al., each of which is incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under contractR43CA133987 awarded by the National Cancer Institute. The government hascertain rights in the invention.

FIELD OF THE INVENTION

Described are systems and methods for detecting analyte molecules orparticles in a fluid sample and in some cases, determining a measure ofthe concentration of the molecules or particles in the fluid sample.

BACKGROUND OF THE INVENTION

Methods and systems that are able to quickly and accurately detect and,in certain cases, quantify a target analyte molecule in a sample are thecornerstones of modern analytical measurements. Such systems and/ormethods are employed in many areas such as academic and industrialresearch, environmental assessment, food safety, medical diagnosis, anddetection of chemical, biological, and/or radiological warfare agents.Advantageous features of such techniques may include specificity, speed,and sensitivity.

Most current techniques for quantifying low levels of analyte moleculesin a sample use amplification procedures to increase the number ofreporter molecules in order to be able to provide a measurable signal.For example, these known processes include enzyme-linked immunosorbentassays (ELISA) for amplifying the signal in antibody-based assays, aswell as the polymerase chain reaction (PCR) for amplifying target DNAstrands in DNA-based assays. A more sensitive but indirect proteintarget amplification technique, called immunoPCR (see Sano, T.; Smith,C. L.; Cantor, C. R. Science 1992, 258, 120-122), makes use ofoligonucleotide markers, which can subsequently be amplified using PCRand detected using a DNA hybridization assay (see Nam, J. M.; Thaxton,C. S.; Mirkin, C. A. Science 2003; 301, 1884-1886; Niemeyer, C. M.;Adler, M.; Pignataro, B.; Lenhert, S.; Gao, S.; Chi, L. F.; Fuchs, H.;Blohm, D. Nucleic Acids Research 1999, 27,4553-4561; and Zhou, H.;Fisher, R. J.; Papas, T. S. Nucleic Acids Research 1993, 21, 6038-6039).While the immuno-PCR method permits ultra low-level protein detection,it is a complex assay procedure, and can be prone to false-positivesignal generation (see Niemeyer, C. M.; Adler, M.; Wacker, R. Trends inBiotechnology 2005, 23,208-216).

One feature of typical known methods and/or systems for detecting orquantifying low concentrations of a particular analyte in solution isthat they are based on ensemble responses in which many analytemolecules give rise to a measured signal. Most detection schemes requirethat a large number of molecules are present in the ensemble for theaggregate signal to be above the detection threshold. This requirementlimits the sensitivity of most detection techniques and the dynamicrange (i.e., the range of concentrations that can be detected). Many ofthe known methods and techniques are further plagued with problems ofnon-specific binding, which is the binding of analyte molecules orparticles to be detected or reporter species non-specifically to sitesother than those expected. This leads to an increase in the backgroundsignal, and therefore limits the lowest concentration that may beaccurately or reproducibly detected.

Accordingly, improved methods for detecting and, optionally, quantifyinganalyte molecules or particles in a fluid sample are needed, especiallyin samples where such molecules or particles are present at very lowconcentration.

SUMMARY OF THE INVENTION

Described herein are systems and methods for detecting analyte moleculesor particles in a fluid sample and in some cases, determining a measureof the concentration of the molecules or particles in the fluid sample.The subject matter of the present invention involves, in some cases,interrelated products, alternative solutions to a particular problem,and/or a plurality of different uses of one or more systems and/orarticles.

In some embodiments, a method for determining a measure of theconcentration of analyte molecules or particles in a fluid samplecomprises exposing a plurality of capture objects that each include abinding surface having affinity for at least one type of analytemolecule or particle, to a solution containing or suspected ofcontaining the at least one type of analyte molecules or particles,immobilizing analyte molecules or particles with respect to theplurality of capture objects such that at least some of the captureobjects associate with a single analyte molecule or particle and astatistically significant fraction of the capture objects do notassociate with any analyte molecule or particle, spatially segregatingat least a portion of the capture objects subjected to the immobilizingstep into a plurality of separate locations, addressing at least aportion of the plurality of locations subjected to the spatiallysegregating step and determining the number of said locations containingan analyte molecule or particle, and determining a measure of theconcentration of analyte molecules or particles in the fluid samplebased at least in part on the number of locations determined to containan analyte molecule or particle.

In some embodiments, a method for determining a measure of theconcentration of analyte molecules or particles in a fluid samplecomprises exposing a plurality of capture objects that each include abinding surface having affinity for at least one type of analytemolecule or particle, to a solution containing or suspected ofcontaining the at least one type of analyte molecules or particles toform capture objects comprising at least one immobilized analytemolecule or particle, mixing the capture objects prepared in theexposing step to a plurality of binding ligands such that at least someof the capture objects associate with a single binding ligand and astatistically significant fraction of the capture objects do notassociate with any binding ligand, spatially segregating at least aportion of the capture objects subjected to the mixing step into aplurality of locations, addressing at least a portion of the pluralityof locations subjected to the spatially segregating step and determiningthe number of locations containing a binding ligand, and determining ameasure of the concentration of analyte molecules or particles in thefluid sample based at least in part on the number of locationsdetermined to contain a binding ligand.

In some embodiments, a method for determining a measure of theconcentration of analyte molecules or particles in a fluid samplecomprises providing a substrate comprising a plurality of locations, atleast a portion of which locations contain a bead, wherein with respectto the total number of beads present on the substrate, the ratio ofbeads comprising at least one analyte molecule or particle to beadscomprising no analyte molecules or particles is between about 8:1 andabout 1:10,000,000, addressing at least a portion of the plurality oflocations, wherein during the addressing step at least two of theplurality of locations is addressed at least partially concurrently,detecting at each addressed location the presence or absence of a beadand whether, if present, the bead comprises any analyte molecules orparticles, and determining a measure of the concentration of analytemolecules or particles in the fluid sample at least in part bydetermining the number of locations addressed containing a beadcomprising at least one analyte molecule or particle.

In some embodiments, a method for determining a measure of theconcentration of analyte molecules or particles in a fluid samplecomprises providing a substrate comprising a plurality of locations, atleast a portion of which contain a bead, wherein with respect to thetotal number of beads present on the substrate, the ratio of beadscomprising at least one analyte molecule or particle associated with abinding ligand to beads comprising no analyte molecules or particlesassociated with a binding ligand is between about 8:1 and about1:10,000,000, addressing at least a portion of the plurality oflocations, wherein during the addressing step at least two of theplurality of locations is addressed at least partially concurrently,detecting at each addressed location the presence or absence of a beadand whether, if present, the bead comprises any analyte molecules orparticles associated with a binding ligand, and determining a measure ofthe concentration of analyte molecules or particles in the fluid sampleat least in part by determining the number of locations addressedcontaining a bead comprising at least one analyte molecule or particleassociated with a binding ligand.

In some embodiments, an article or kit comprises a plurality of beadshaving an average diameter between about 0.1 micrometer and about 100micrometers, and a substrate comprising a plurality of reaction vessels,wherein the average depth of the reaction vessels is between about 1.0times and about 1.5 times the average diameter of the beads and theaverage diameter of the reactions vessels is between about 1.0 times andabout 1.9 times the average diameter of the beads.

In some embodiments, a method for determining a measure of theconcentration of analyte molecules or particles in a fluid samplecomprises exposing a plurality of capture objects that each include abinding surface having affinity for at least one type of analytemolecule or particle, to a solution containing or suspected ofcontaining the at least one type of analyte molecules or particles,wherein at least some of the capture objects become associated with atleast one analyte molecule or particle, mixing the plurality of captureobjects prepared in the exposing step to a plurality of binding ligandscomprising an enzymatic component such that a statistically significantfraction of the capture objects associated with at least one analytemolecule or particle associate with a single binding ligand, spatiallysegregating at least a portion of the capture objects subjected to themixing step into a plurality of separate locations, determining ameasure of the concentration of analyte molecules or particles in thefluid sample based at least in part by addressing at least a portion ofthe plurality of locations subjected to the spatially segregating stepto determine the presence of the enzymatic component or a product of areaction involving the enzymatic component.

In some embodiments, a method for determining a measure of theconcentration of analyte molecules or particles in a fluid samplecomprises immobilizing a plurality of analyte molecules or particleswith respect to a plurality of beads, spatially segregating at least aportion of the plurality of beads into a plurality of separatelocations, and addressing at least some of the plurality of locationsand determining the number of locations containing a bead, and furtherdetermining the number of said locations containing a bead and ananalyte molecule or particle, and determining a measure of theconcentration of analyte molecules or particles in the fluid samplebased at least in part on the ratio of the number of locationscontaining a bead and an analyte molecule and particle, to the number oflocations containing a bead.

In some embodiments, a method for determining a measure of theconcentration of analyte molecules or particles in a fluid samplecomprises immobilizing a plurality of analyte molecules or particleswith respect to a plurality of beads, spatially segregating at least aportion of the plurality of beads into a plurality of separatelocations, addressing at least some of the plurality of locations anddetermining the number of locations containing a bead, furtherdetermining the number of said locations containing a bead and ananalyte molecule or particle, and determining a measure of theconcentration of analyte molecules or particles in the fluid samplebased at least in part on the ratio of the number of locationscontaining a bead and an analyte molecule and particle, to the number oflocations containing a bead but not containing any analyte molecules orparticles.

In some embodiments, a method for determining a measure of theconcentration of analyte molecules or particles in a fluid samplecomprises providing a plurality of capture objects that each areassociated with either a single analyte molecule or particle or are freeof any analyte molecules or particles, individually addressing at leasta portion of the capture objects and determining the number of saidcapture objects associated with an analyte molecule or particle, anddetermining a measure of the concentration of analyte molecules orparticles in the fluid sample based at least in part on the number ofcapture objects subjected to the addressing step determined to beassociated with an analyte molecule or particle.

BRIEF DESCRIPTION OF THE DRAWINGS

Other aspects, embodiments, and features of the invention will becomeapparent from the following detailed description when considered inconjunction with the accompanying drawings. The accompanying figures areschematic and are not intended to be drawn to scale. For purposes ofclarity, not every component is labeled in every figure, nor is everycomponent of each embodiment of the invention shown where illustrationis not necessary to allow those of ordinary skill in the art tounderstand the invention. All patent applications and patents mentionedin the text are incorporated by reference in their entirety. In case ofconflict between the description contained in the present specificationand a document incorporated by reference, the present specification,including definitions, will control.

FIG. 1 is a schematic flow diagram depicting one embodiment of steps(A-D) for performing an exemplary method of the present invention;

FIG. 2 is a schematic flow diagram depicting one embodiment of steps(A-D) for performing an exemplary method of the present invention;

FIG. 3 is a schematic diagram depicting one embodiment of a portion of amethod of the present invention;

FIG. 4A is a schematic flow diagram depicting one embodiment of steps(A-C) for performing an exemplary method of the present invention;

FIG. 4B is a schematic flow diagram depicting one embodiment of steps(A-D) for performing an exemplary method of the present invention;

FIG. 4C is a schematic diagram depicting one embodiment for performingan exemplary method of the present invention;

FIG. 5 is a schematic flow diagram depicting one embodiment of steps(A-C) for performing an exemplary method of the present invention;

FIG. 6 is a schematic flow diagram depicting an embodiment of a method(steps A-D) for the formation of a plurality of reaction vessels throughmating of a substrate and a sealing component and depicting examples ofthe size (E, F) of a sealing component relative to a substrate;

FIG. 7 depicts an experimental set-up for detection using light,according to one embodiment of the present invention;

FIG. 8 shows a fiber optic array that has been sealed with a sealingcomponent, according to one embodiment;

FIG. 9A shows a schematic diagram depicting a method of indirectlydetecting an analyte molecule associated with a capture object,according to some embodiments;

FIG. 9B shows a schematic diagram depicting a method of indirectlydetecting an analyte molecule immobilized with respect to a captureobject using a binding ligand, according to some embodiments;

FIG. 10A and FIG. 10B show non-limiting examples of a system employingan optical detection system of the present invention according to someembodiments;

FIG. 11 is a schematic block diagram showing a system employing a fiberoptic assembly with an optical detection system according to anembodiment of the invention;

FIG. 12 shows a graph of a schematic calibration curve that may be usedto determine the concentration of analyte molecules or particles in afluid sample, according to some embodiments of the present invention;

FIG. 13 shows a graph of the number of reaction vessels comprising abead versus the total number of beads provided to the reaction vessels,according to a non-limiting embodiment;

FIG. 14A, FIG. 14B and FIG. 14C show non-limiting images of beadscontained in arrays comprising a plurality of reaction vessels;

FIG. 15A shows a non-limiting fluorescence image of an array containingbeads,

FIG. 15B shows an enlargement of the image from FIG. 15A;

FIG. 16A and FIG. 16B show graphs of the number of reaction vesselsdetermined to contain an analyte molecule versus the concentration ofanalyte molecules in a fluid sample, according to certain embodiments;

FIG. 16C shows a graph of the total fluorescence read-out versus theconcentration of analyte molecules in a fluid sample, according to anexemplary embodiment;

FIG. 17 shows a plot of the % Poisson Noise against the experimentalvariance over three measurements from the experimental data shown inFIG. 16B.

FIG. 18 shows a plot of the fraction of capture objects determined to beassociated with an analyte molecule versus the concentration of bindingligand provided, at two concentrations of analyte molecules, accordingto an exemplary embodiment;

FIG. 19 shows a plot of the fraction of capture objects determined to beassociated with an analyte molecule versus the concentration of bindingligand per capture object provided, at two concentrations of analytemolecules, according to an exemplary embodiment;

FIG. 20 shows a plot of the fraction of capture objects determined to beassociated with an analyte molecule versus the concentration of bindingligand provided, at two concentrations of analyte molecules, accordingto an exemplary embodiment;

FIG. 21 shows a plot of the total chemiluminescence versus theconcentration of binding ligand provided, according to an exemplaryembodiment;

FIG. 22 shows a plot of the log of the fraction of capture objectsdetermined to be associated with an analyte molecule versus the log ofthe concentration of binding ligand provided, according to an exemplaryembodiment;

FIG. 23A and FIG. 23B show schematic diagrams depicting one embodimentof steps for performing one method of the present invention;

FIG. 23C shows an image of beads contained in a plurality of reactionvessels, according to an exemplary embodiment;

FIG. 23D shows a fluorescence image of an array comprising a pluralityof beads, some of which are associated with an analyte moleculefollowing carrying out a method of the present invention, according toan exemplary embodiment.

FIG. 24 shows a plot of the log of the fraction of capture objectsdetermined to be associated with an analyte molecule versus the log ofthe concentration of analyte molecules in a fluid sample, according toan exemplary embodiment;

FIG. 25A shows plots of the fraction of capture objects determined to beassociated with an analyte molecule comprising PSA, versus theconcentration of analyte molecules in a fluid sample, according to anexemplary embodiment;

FIG. 25B shows plots of the fraction of capture objects determined to beassociated with an analyte molecule comprising TNF-alpha, versus theconcentration of analyte molecules in a fluid sample, according to anexemplary embodiment;

FIG. 25C shows plots of the fraction of capture objects determined to beassociated with an analyte molecule comprising DNA, versus theconcentration of analyte molecules in a fluid sample, according to anexemplary embodiment;

FIG. 26 shows a plot of the optical density versus the concentration ofTNF-alpha, according to an exemplary embodiment;

FIG. 27 shows a plot of the concentration of PSA determined for aplurality of human subjects; and

FIG. 28 shows a histogram of the average fluorescence intensity ofreaction vessels in an assay method, according to one embodiment of thepresent invention.

DETAILED DESCRIPTION

Described herein are systems and methods that may in certain embodimentsbe employed for the detection and/or quantification of analytemolecules, particles (such as, for example, cells, cell organelles andother biological or non-biological particulates), and the like, in asample. The subject matter of the present invention involves, in somecases, interrelated products, alternative solutions to a particularproblem, and/or a plurality of different uses of one or more systemsand/or articles. It should be understood, that while much of thediscussion below is directed to analyte molecules, this is by way ofexample only, and other materials may be detected and/or quantified, forexample, analytes in particulate form. Some exemplary analyte moleculesand particles are described herein.

The systems and methods of the present invention in certain instancesmay help reduce the negative effects of non-specific binding ondetection sensitivity when compared to typical conventional systems andmethods for performing similar assays. Non-specific binding is thebinding or association in a non-specific fashion of one component of anassay with another component of the assay with which it is not desirablethat it interact. For example, association, binding, or immobilizationof a binding ligand with a substrate or assay material as opposed towith an analyte molecule or particle to which it has bindingspecificity. Non-specific binding may lead to false positive signals.Non-specific binding may not only affect the accuracy of the assaymeasurement, but may also limit the lowest level of detection.Therefore, certain methods and/or systems of the present invention thatprovide improvements in the level of non-specific binding, may allow forthe detection and/or quantification of analyte molecules in a sample ata lower detection limit as compared to typical conventionaltechnologies. In addition, certain embodiments of the methods and/orsystems of the present invention may also allow for the detection and/orquantification of analyte molecules in certain samples in which suchanalyte molecules have previously been undetected and/or unquantifiablebecause of the very low concentration in which they are present.

Certain methods of the present invention may be useful forcharacterizing analyte molecules in a sample. In some cases, the methodsmay be useful for detecting and/or quantifying analyte molecules in afluid sample which is suspected of containing at least one type ofanalyte molecule, since, as explained in more detail below, theinventive assays may be designed such that the number (or equivalentlyfraction) of interrogated locations (e.g., wells, reaction sites, areason a surface, etc.) which contain a capture object (e.g., bead, surface,etc. providing a capture surface) comprising an analyte molecule—or,more generally, the number or fraction of interrogated capture objectsof a total interrogated population comprising an analyte molecule—can becorrelated to the concentration of analyte molecules in the fluidsample. Certain embodiments of present invention thus can provide ameasure of the concentration of analyte molecules in a fluid samplebased at least in part on the number or fraction of locations, e.g., ona substrate, which contain a capture object associated with an analytemolecule. In some cases, this number/fraction may be related to thetotal number of locations comprising a capture object (e.g., with orwithout an associated analyte molecule or labeling agent) and/or to thetotal number of locations interrogated. Specific methods andcalculations of how to quantify analyte molecules in a fluid sampleusing embodiments of the invention are discussed more below.

In certain embodiments, a method for detection and/or quantifyinganalyte molecules (or particles) in a sample comprises immobilizing aplurality of analyte molecules with respect to a plurality of captureobjects that each include a binding surface having affinity for at leastone type of analyte molecule (or particle). For example, the captureobjects may comprise a plurality of beads comprising a plurality ofcapture components (e.g., an antibody having specific affinity for ananalyte molecule of interest, etc.). At least some of the captureobjects (e.g., at least some associated with at least one analytemolecule) may be spatially separated/segregated into a plurality oflocations, and at least some of the locations may beaddressed/interrogated. A measure of the concentration of analytemolecules in the fluid sample may be determined based on the informationreceived when addressing the locations. In some cases, a measure of theconcentration may be based at least in part on the number of locationsdetermined to contain a capture object that is or was associated with atleast one analyte molecule. In other cases and/or under differingconditions, a measure of the concentration may be based at least in parton an intensity level of at least one signal indicative of the presenceof a plurality of analyte molecules and/or capture objects associatedwith an analyte molecule at one or more of the addressed locations.

In some embodiments, the number/fraction of locations containing acapture object but not containing an analyte molecule may also bedetermined and/or the number/fraction of locations not containing anycapture object may also be determined.

In such embodiments, a measure of the concentration of analyte moleculein the fluid sample may be based at least in part on the ratio of thenumber of locations determined to contain a capture object associatedwith an analyte molecule to the total number of locations determined tocontain a capture object not associated with an analyte molecule and/ora measure of the concentration of analyte molecule in the fluid samplemay be based at least in part on the ratio of the number of locationsdetermined to contain a capture object associated with an analytemolecule to the number of locations determined to not contain anycapture objects. In yet other embodiments, a measure of theconcentration of analyte molecules in a fluid sample may be based atleast in part on the ratio of the number of locations determined tocontain a capture object and an analyte molecule to the total number oflocations addressed and/or analyzed.

In certain embodiments, at least some of the plurality of captureobjects (e.g., at least some associated with at least one analytemolecule) are spatially separated into a plurality of locations, forexample, a plurality of reaction vessels in an array format. Theplurality of reaction vessels may be formed in, on and/or of anysuitable material, and in some cases, the reaction vessels can be sealedor may be formed upon the mating of a substrate with a sealingcomponent, as discussed in more detail below. In certain embodiments,especially where quantization of the capture objects associated with atleast one analyte molecule is desired, the partitioning of the captureobjects can be performed such that at least some (e.g., a statisticallysignificant fraction) of the reaction vessels comprise at least one or,in certain cases, only one capture object associated with at least oneanalyte molecule and at least some (e.g., a statistically significantfraction) of the reaction vessels comprise an capture object notassociated with any analyte molecules. The capture objects associatedwith at least one analyte molecule may be quantified in certainembodiments, thereby allowing for the detection and/or quantification ofanalyte molecules in the fluid sample by techniques described in moredetail herein.

An exemplary embodiment of an inventive assay method is illustrated inFIG. 1. A plurality of capture objects 2, are provided (step (A)). Inthis particular example, the plurality of capture objects comprises aplurality of beads. The beads are exposed to a fluid sample containing aplurality of analyte molecules 3 (e.g., beads 2 are incubated withanalyte molecules 3). At least some of the analyte molecules areimmobilized with respect to a bead. In this example, the analytemolecules are provided in a manner (e.g., at a concentration) such thata statistically significant fraction of the beads associate with asingle analyte molecule and a statistically significant fraction of thebeads do not associate with any analyte molecules. For example, as shownin step (B), analyte molecule 4 is immobilized with respect to bead 5,thereby forming complex 6, whereas some beads 7 are not associated withany analyte molecules. It should be understood, in some embodiments,more than one analyte molecule may associate with at least some of thebeads, as described herein. At least some of the plurality of beads(e.g., those associated with a single analyte molecule or not associatedwith any analyte molecules) may then be spatially separated/segregatedinto a plurality of locations. As shown in step (C), the plurality oflocations is illustrated as substrate 8 comprising a plurality ofwells/reaction vessels 9. In this example, each reaction vesselcomprises either zero or one beads. At least some of the reactionvessels may then be addressed (e.g., optically or via other detectionmeans) to determine the number of locations containing an analytemolecule. For example, as shown in step (D), the plurality of reactionvessels are interrogated optically using light source 15, wherein eachreaction vessel is exposed to electromagnetic radiation (represented byarrows 10) from light source 15. The light emitted (represented byarrows 11) from each reaction vessel is determined (and/or recorded) bydetector 15 (in this example, housed in the same system as light source15). The number of reaction vessels containing an analyte molecule(e.g., reaction vessels 12) is determined based on the light detectedfrom the reaction vessels. In some cases, the number of reaction vesselscontaining a bead not associated with an analyte molecule (e.g.,reaction vessel 13), the number of wells not containing a bead (e.g.,reaction vessel 14) and/or the total number of wells addressed may alsobe determined. Such determination(s) may then be used to determine ameasure of the concentration of analyte molecules in the fluid sample.

A statistically significant fraction of capture objects that contain atleast one analyte molecule (or no analyte molecules) will typically beable to be reproducibly detected and quantified using a particularsystem of detection and will typically be above the background noise(e.g., non-specific binding) that is determined when carrying out theassay with a sample that does not contain any analyte molecules, dividedby the total number of objects (or locations) addressed. A“statistically significant fraction” as used herein for the presentembodiments, may be estimated according to the Equation 1:

n≧3√{square root over (n)}  (Eq. 1)

wherein n is the number of determined events for a selected category ofevents. That is, a statistically significant fraction occurs when thenumber of events is greater than three times square root of the numberof events. For example, to determine a statistically significantfraction of the capture objects not associated with any analytemolecules or particles, n is the number of capture objects detected thatare not associated with any analyte molecules or particles. As anotherexample, to determine a statistically significant fraction of thecapture objects associated with at least one analyte molecule, n is thenumber of capture objects detected that are determined to be associatedwith an analyte molecule.

In some embodiments, the statistically significant fraction of captureobjects (e.g., beads) associated with at least one analyte molecule (ora single analyte molecule in some cases where the ratio of mixingcapture objects to analyte molecules would lead, statistically, to onlyzero or one analyte molecule associate with each capture object) to thetotal number of capture objects (e.g., beads) is less than about 1:2,less than about 1:3, less than about 1:4. is less than about 2:5, lessthan about 1:5, less than about 1:10, less than about 1:20, less thanabout 1:100, less than about 1:200, or less than about 1:500. Therefore,in such embodiments, the fraction of capture objects (e.g., beads) notassociated with any analyte molecules to the total number of captureobjects (e.g., beads) is at least about 1:100, about 1:50, about 1:20,about 1:10, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about2:1, about 3:1, about 4:1, about 5:1, about 10:1, about 20:1, about50:1, about 100:1, or the like.

In some embodiments, the percentage of capture objects (e.g., beads)associated with at least one analyte molecule (or a single analytemolecule in some cases where the ratio of mixing capture objects toanalyte molecules would lead, statistically, to only zero or one analytemolecule associate with each capture objects) is less than about 50%,less than about 40%, less than about 30%, less than about 20%, less thanabout 10%, less than about 5%, less than about 2%, less than about 1%,less than about 0.5%, less than about 0.01%, or the like, the totalnumber of capture objects. In some embodiments, the percentage ofcapture objects (e.g., beads) not associated with an analyte molecule tothe total number of capture objects (e.g., beads) is at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 98%, or the like, the total number of capture objects.

In some embodiments, prior to spatially separating the plurality ofcapture objects, the capture objects may be exposed to a plurality ofbinding ligands which have an affinity for at least one type of analytemolecule (or particle). A “binding ligand,” as used herein, is anymolecule, particle, or the like which specifically binds to or otherwisespecifically associates with an analyte molecule to aid in the detectionof the analyte molecule. Binding ligands may be particularly useful inembodiments where at least some of the capture objects are associatedwith respect to more than one analyte molecule (e.g., two, three, four,five, or more, analyte molecules). In some cases, the binding ligand maybe provided in a manner (e.g., at a concentration level) such that astatistically significant fraction of the capture objects comprising atleast one analyte molecule associate with at least one binding ligand(or in some cases, a single binding ligand) and a statisticallysignificant fraction of the capture objects (e.g., capture objectseither associated with at least one analyte molecule or not associatedwith any analyte molecules) do not associate with any binding ligand.

A statistically significant fraction of the locations that contain acapture object (e.g., bead) associated with at least one analytemolecule and a single binding ligand is greater than or equal to theminimum number of locations that can be reproducibly determined tocontain an capture object (e.g., bead) associated with a single bindingligand with a particular system of detection (i.e., substantiallysimilar results are obtained for multiple essentially identical fluidsamples comprising the capture objects associated with an analytemolecule and/or binding ligand) and that is above the background noise(e.g., non-specific binding) that is determined when carrying out theassay with a sample that does not contain any analyte molecules and/orbinding ligands, divided by the total number of locations. Thestatistically significant fraction of locations that contain a captureobject associated with at least one analyte molecule and a singlebinding ligand can be determined according to Equation 1. The ratio ofthe number of capture objects to analyte molecules and/or bindingligands which may be provided such that substantially all of the captureobjects are associated with zero or a single analyte molecule may becalculated using a Poisson distribution adjustment, as described herein.

In some embodiments, the statistically significant fraction of captureobjects (e.g., beads) associated with at least one analyte molecule andat least one binding ligand to the total number of capture objects(e.g., beads) is less than about 1:2, less than about 1:3, less thanabout 1:4. is less than about 2:5, less than about 1:5, less than about1:10, less than about 1:20, less than about 1:100, less than about1:200, or less than about 1:500. In some cases, the statisticallysignificant fraction of capture objects (e.g., beads) associated notassociated with any binding ligand to the total number of captureobjects at least about 1:100, about 1:50, about 1:20, about 1:10, about1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1,about 4:1, about 5:1, about 10:1, about 20:1, about 50:1, about 100:1,or the like.

In some embodiments, the percentage of capture objects (e.g., beads)associated with at least one analyte molecule and at least one bindingligand to the total number of capture objects (e.g., beads) is less thanabout 50%, less than about 40%, less than about 30%, less than about20%, less than about 10%, less than about 5%, less than about 2%, lessthan about 1%, less than about 0.5%, less than about 0.01%, or less. Insome embodiments, the percentage of capture objects (e.g., beads) notassociated with any binding ligand to the total number of captureobjects is at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, at least about 98%, or greater.

A non-limiting example of an embodiment where a capture object isassociated with more than one analyte molecule is illustrated in FIG. 2.A plurality of capture objects 20 are provided (step (A)). In thisexample, the plurality of capture objects comprises a plurality ofbeads. The plurality of beads is exposed to a fluid sample containingplurality of analyte molecules 21 (e.g., beads 20 are incubated withanalyte molecules 21). At least some of the analyte molecules areimmobilized with respect to a bead. For example, as shown in step (B),analyte molecule 22 is immobilized with respect to bead 24, therebyforming complex 26. Also illustrated is complex 30 comprising a beadimmobilized with respect to three analyte molecules and complex 32comprising a bead immobilized with respect to two analyte molecules.Additionally, in some cases, some of the beads may not associate withany analyte molecules (e.g., bead 28). The plurality of beads from step(B) is exposed to a plurality of binding ligands 31. As shown in step(C), a binding ligand associates with some of the analyte moleculesimmobilized with respect to a bead. For example, complex 40 comprisesbead 34, analyte molecule 36, and binding ligand 38. The binding ligandsare provided in a manner such that a statistically significant fractionof the beads comprising at least one analyte molecule become associatedwith at least one binding ligand (e.g., one, two, three, etc.) and astatistically significant fraction (i.e. as determined by Equation 1above) of the beads comprising at least one analyte molecule do notbecome associated with any binding ligands. At least a portion of theplurality of beads from step (C) are then spatially separated into aplurality of locations. As shown in step (D), in this example, thelocations comprise a plurality of reaction vessels 41 on a substrate 42.The plurality of reaction vessels may be exposed to the plurality ofbeads from step (C) such at each reaction vessel contains zero or onebeads. The substrate may then be analyzed to determine the number ofreaction vessels containing a binding ligand (e.g., reaction vessels43), wherein in the number may be related to a measure of theconcentration of analyte molecules in the fluid sample. In some cases,the number of reaction vessels containing a bead and not containing abinding ligand (e.g., reaction vessel 44), the number of reactionvessels not containing a bead (e.g., reaction vessel 45), and/or thetotal number of reaction vessels addressed/analyzed may also bedetermined. Such determination(s) may then be used to determine ameasure of the concentration of analyte molecules in the fluid sample.

The foregoing exemplary methods may be performed using a number ofdifferent assay formats, different reaction conditions, and/or detectionsystems in different embodiments of the invention, several examples ofwhich are described below. Additional components and/or method steps maybe utilized as a substitute for and/or in combination with the exemplarymethods and components described herein within the scope of theinvention. It should be understood, while certain of the discussionherein focuses on a plurality of locations comprising a plurality ofwells/reaction vessels in a substrate, this is by no means limiting andother materials may be used to segregate capture objects/molecules intoa plurality of spatially distinct locations (e.g., regions in/on ahydrogel, points/regions on the surface of a planar substrate, etc.). Asanother example, while much of the discussion herein focuses on aplurality of capture objects comprising a plurality of beads, this is byno means limiting and in other embodiments the capture objects may takeother physical forms (e.g., nanotubes, disks, rings, microfluidicdroplets, etc.).

Exemplary Assay Formats

The inventive assays may be carried our according to a very wide varietyof basic protocols and formats. The particular format chosen can bebased on the nature of the analyte molecules, the nature of the fluidsample containing the analyte molecules, and the availability andproperties of binding partners of the analyte as well as other factors.Several exemplary basic formats were discussed previously in the contextof the discussion of FIGS. 1-2. As would be apparent to those skilled inthe art with the benefit of the teachings provided by the presentdisclosure, the invention may alternatively be performed according toprotocols/formats not specifically described in the specific, exemplaryembodiments illustrated in this detailed description, but which do notrequire undue burden or experimentation to practice.

As described above, an exemplary basic assay format/protocol comprisesexposing a plurality of capture objects (e.g., beads) configured tocapture an analyte molecule or particle to a sample containing orsuspected of containing such analyte molecules (or particles). At leastsome of the analyte molecules may become immobilized with respect to acapture object. The plurality of capture objects may each include abinding surface having affinity for at least one type of analytemolecule. At least a portion of the capture objects may then bespatially segregated into a plurality of locations (e.g., reactionvessels/wells). Based at least in part on a determination of the numberof locations comprising a capture object comprising at least one analytemolecule, a measure of the concentration of analyte molecules may bedetermined. Various other aspects of this basic assay format will now bediscussed, including numerous considerations regarding the materials,concentrations, solutions, steps, and the like.

In certain embodiments, a plurality of capture objects is exposed to asample containing or suspected of containing at least one type ofanalyte molecules, wherein the plurality of capture objects comprises abinding surface having an affinity for the at least one type of analytemolecule. In some cases, the binding surface may comprise a plurality ofcapture components. A “capture component”, as used herein, is anymolecule, other chemical/biological entity, or solid supportmodification disposed upon a solid support that can be used tospecifically attach, bind or otherwise capture a target molecule orparticle (e.g., an analyte molecule), such that the targetmolecule/particle becomes immobilized with respect to the capturecomponent and the support. The immobilization, as described herein, maybe caused by the association of an analyte molecule with a capturecomponent on the surface of the capture object. As used herein,“immobilized” means captured, attached, bound, or affixed so as toprevent dissociation or loss of the target molecule/particle, but doesnot require absolute immobility with respect to either the capturecomponent or the object.

The number of analyte molecules which are immobilized with respect to acapture object may depend on the ratio of the total number of analytemolecules in the sample versus at least one of the total number, size,and/or surface density of capture components of capture objectsprovided. In some embodiments, the number of molecules or particlesimmobilized with respect to a single capture object may follow astandard Poisson distribution. In some cases, a statisticallysignificant number of the capture objects associate with a singleanalyte molecule and a statistically significant number of captureobjects do not associate with any analyte molecules. The total number ofcapture objects provided may be between about 10,000 and about10,000,000, between about 50,000 and about 5,000,000, or between about100,000 and about 1,000,000. In some cases, the total number of captureobjects provided is at least about 10,000, at least about 50,000, atleast about 100,000, at least about 1,000,000, at least about 5,000,000,or at least about 10,000,000. In some cases, the ratio of the number ofanalyte molecules in the fluid sample to capture objects provided isbetween about 10:1 and about 1:10,000,000, between about 8:1 and about1:10,000,000, between about 10:1 and about 2:1, between about 2:1 andabout 1:10, or less than about 1:10 (e.g., about 1:20, about 1:30,etc.). The ratio of analyte molecules in the fluid sample to captureobjects provided may affect the assay steps and/or analysis carried outto determine a measure of the concentration of analyte molecules in thefluid sample, as described herein in the Quantification section.

In some cases, substantially all of the analyte molecules provided inthe sample may become immobilized with respect to a capture object. Thatis, greater than about 90%, greater than about 95%, greater than about97%, greater than about 98%, or greater than about 99% of the analytemolecules in the sample may become immobilized with respect to a captureobject. In some cases, however, only a fraction of the analyte moleculesin the sample may become immobilized with respect to a capture object.That is, in some cases, between about 1% and about 90%, between about10% and about 90%, between about 20% and about 80%, or between about 30%and about 70% of the analyte molecules provided in the sample areimmobilized with respect to a capture object. In some embodiments, atleast about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, or about 90%, or about 95% of the analytemolecules are immobilized with respect to a capture object.

In some formats of the assay, following immobilization, the plurality ofcapture objects (e.g., at least some of which are associated with atleast one analyte molecule) may be exposed to a plurality of bindingligands. At least some of the analyte molecules immobilized with respectto a capture object may associate with a binding ligand. The number ofbinding ligands which associate with a capture object (e.g., via ananalyte molecule) may depend on the ratio of the total number of analytemolecules immobilized with respect to a single capture object versus thetotal number of binding ligands exposed to the capture objects. Forexample, in embodiments where substantially all of the capture objectsare associated with either zero or one analyte molecules, conditions maybe selected such that substantially all of the analyte moleculesassociate with a single binding ligand, therefore each capture objectassociated with a single analyte molecule becomes associated with asingle binding ligand (e.g., via the analyte molecule). Thus, the numberof locations (e.g., reaction vessels) which contain a single analytemolecule may be determined by determining the number of locations (e.g.,reaction vessels) which comprise a binding ligand. In such embodiments(e.g., where zero or at least one analyte molecules are associated witheach capture object), the ratio of binding ligands provided (e.g., in amixing step) to the total number of analyte molecules immobilized withrespect to a capture object may be about 20:1, about 10:1, about 5:1,about 2:1, or about 1:1.

In some embodiments, however, a single capture object may be associatedwith zero, one, or more than one (e.g., two, three, four, etc.) analytemolecules. In such embodiments, the binding ligand may be provided at aconcentration such that a statistically significant fraction of thecapture objects comprising at least one analyte molecule associate withonly a single binding ligand and a statistically significant fraction ofthe capture objects comprising at least one analyte molecule do notassociate with any binding ligand. In other embodiments, however, thebinding ligands may be provided at a concentration such that astatistically significant fraction of the capture objects comprising atleast one analyte molecule associate with at least one binding ligand(e.g., one, two, three, etc.) and a statistically significant fractionof the capture objects comprising at least one analyte molecule do notassociate with any binding ligand. The concentration of analytemolecules in the fluid sample may then be determined, either with ananalysis based at least in part of the number of locations containing acapture object associated with a binding ligand (e.g., by relating theconcentration of analyte molecules in the fluid sample to the number oflocations comprising a binding ligand), and/or an analysis based atleast in part on an intensity reading of a signal indicative of thenumber of binding ligands at the addressed locations (e.g., inembodiments where at least some of the capture objects comprise morethan one analyte molecule and/or more than one binding ligand, asdescribed herein). In such embodiments (e.g., wherein more than oneanalyte molecule may be immobilized with respect to each captureobject), the ratio of the number of binding ligands provided in solutionto the number of analyte molecules immobilized with respect to a captureobject may be about 1:50, about 1:40, about 1:30, about 1:20, about1:10, about 1:5, about 1:3, about 1:2, about 1:1, or the like. In somecases, the ratio of the number of binding ligands provided in solutionmay be calculated based on the number of capture objects provided. Insome cases, the ratio of binding ligands provided to the number ofcapture objects is about 1:50, about 1:40, about 1:30, about 1:20, about1:10, about 1:5, about 1:3, about 1:2, about 1:1, or the like. In othercases, the ratio the number of capture objects to the number of bindingligands provided is about 1:50, about 1:40, about 1:30, about 1:20,about 1:10, about 1:5, about 1:3, about 1:2, or the like. In someembodiments, the quantification determination may comprise a Poissondistribution adjustment, as described herein.

In some embodiments, the concentration of binding ligand used in anassay may be selected as to minimize certain events which may occur whenan excess of binding ligand is present, for example, non-specificbinding of the binding ligand. In some cases, if the concentration ofbinding ligand is too high, an increase in background readings may occurdue to non-specific interactions (e.g., with the capture objects,reaction vessels, etc.). In some cases, the concentration of bindingligand may be selected (or estimated, in the case of an unknownconcentration of analyte molecule) such that a only a fraction of theanalyte molecules immobilized with respect to a capture object associatewith a binding ligand (e.g., about 0.1%, about 1%, about 2%, about 3%,about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about50%, or more). This may be especially useful in embodiments where thepercentage of capture objects which associate with at least one analytemolecule is relatively high (e.g., greater than about 20%, greater thanabout 30%, greater than about 40%, greater than about 50%, greater thanabout 60%, greater than about 70%, greater than about 80%, greater thanabout 90%, or more). By providing the binding ligand at a lowerconcentration, in some cases, not every analyte molecule immobilizedwith respect to a capture object will associate with a binding ligand,which can be advantageous for quantification, for example when thepresence of a binding ligand is required for detection, and especiallywhen using a digital/binary read-out technique. For example, if thepercentage of capture objects associated with an analyte molecule isabout 50% or greater, a reduced number of binding ligands may beprovided such that less than all of the immobilized analyte moleculesassociate with a binding ligand. In other cases, the percentage ofbinding ligands that associate with an analyte molecule may be reducedby decreasing the incubation time with the analyte molecule (e.g., limitthe time of exposure such that only a fraction of the immobilizedanalyte molecules associate with an analyte molecule).

The total number of analyte molecules/binding ligands/captureobjects/etc. in a solution may be determined using calculations withknowledge of the concentration of the analyte molecules/bindingligands/capture objects/etc. in solution. For example, the total numberof binding ligands in a solution may be determined according to Equation2:

# of binding ligands=N _(A)×[binding ligand]×volume   (Eq. 2)

wherein N_(A) is Avogadro's number (6.022×10²³ mol⁻¹), [binding ligand]is the concentration of the binding ligand in solution in moles perliter, and volume is the total volume of solution in liters employed.Similar calculations may be carried out for other components (e.g.,analyte molecules (e.g., in a calibration sample), capture objects,etc.).

Following immobilization of a plurality of analyte molecules withrespect to a plurality of capture objects and, in some cases,association of a binding ligand to at least some the immobilized analytemolecules, at least a portion of the capture objects may be spatiallysegregated into a plurality of locations. The percentage of captureobjects which are spatially segregated into the plurality of locationsmay vary depending on numerous factors including, but not limited to,the ratio of the number of capture objects versus the total number oflocations, the method of spatially segregating the capture objects,and/or the length of the time the capture objects are exposed to thelocations. In some cases, at least about 0.5%, at least about 1%, atleast about 2%, at least about 5%, at least about 10%, at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least above90%, or more, of the capture objects are spatially segregated into theplurality of locations. In some cases, between about 0.1% and about 50%,between about 0.1% and about 30%, between about 0.5% and about 20%,between about 0.5% and about 10%, between about 0.5% and about 5%,between about 1% and about 10%, or about 0.5%, about 1%, about 2%, about4%, about 5%, about 10%, about 20%, about 30%, about 50%, about 70%, orabout 90% of the capture objects are spatially segregated into theplurality of locations. Following spatially segregating at least aportion of the capture objects into a plurality of locations, at least aportion of the locations may be addressed. The number of locationsaddressed may be about 0.5%, about 1%, about 2%, about 3%, about 5%,about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 90%, about 95%, or more, of the total number oflocations.

The portion of locations may be addressed to determine the number oflocations containing an analyte molecule, or in some cases, a bindingligand. In some cases, the number of locations containing a captureobject not associated with an analyte molecule (or a binding ligand),the number of locations containing and/or not containing a captureobject, and/or the total number of locations analyzed/determined mayalso be determined. A measure of the concentration of analyte moleculesin the fluid sample may be determined at least in part on the number oflocations determined to contain an analyte molecule (or binding ligand).In some cases the measure of the concentration of analyte molecules inthe fluid sample may be based at least in part on the ratio of thenumber of locations containing a capture object associated with ananalyte molecule to the total number of locations addressed or the totalnumber of locations addressed that contain a capture object. In othercases, a measure of the concentration of analyte molecules in the fluidsample may be based at least in part on the ratio of the number oflocations containing a capture object associated with an analytemolecule to the number of locations containing a capture object notassociated with an analyte molecule. Specific methods and calculationswhich may be used to determine the measure of the concentration ofanalyte molecules in the fluid sample are discussed in more detailbelow.

The ratios, percentages, and other parameters described herein withrespect to the amount/quantity/ratio of a first component to a secondcomponent (for example, analyte molecules/capture objects, bindingligands/capture objects, binding ligands/analyte molecules, captureobjects/locations, precursor labeling agents/binding ligands, etc.) maybe adjusted as desired to yield a desired ratio of analytemolecules/binding ligands captured per capture object, and/or may becontrolled or determined using no more than routine experimentation,calculations (in some cases, including accounting for Poissondistributions), screening tests, etc., given the teaching and guidanceprovided by the present specification. For example, if the number ofcapture objects provided is known (e.g., as determined using a similarformula as given in Equation 1), the number of binding ligands that needto be provided may be determined based on the desired ratio of captureobjects to binding ligands, and hence, the amount of moles of bindingligand that should be provided may be determined. As another example, inthe case of an unknown concentration of analyte molecules, if a firstassay method indicates that a significant number of capture objectscomprise more than one analyte molecule (e.g., all or a significantnumber of locations are determined to contain an analyte molecule orthere is less than a statistically significant number of beadsdetermined to be free of analyte molecules), the fluid sample may bediluted and/or the number of capture objects may be increased such thatthe number of capture objects comprising at least one analyte moleculemay be decreased.

Other aspects of the assay will now be discussed in detail. It should beunderstood, that none, a portion of, or all of the following steps maybe performed at least once during the certain exemplary assay formatsdescribed herein. Non-limiting examples of additional steps notdescribed which may be performed include, but are not limited to,washing and/or exposure to additional binding ligands, precursorlabeling agents, and/or labeling agents, etc.

In some embodiments, the plurality of capture objects (e.g., at leastsome of which are associated with at least one analyte molecule) may beexposed to at least one additional reaction component prior to,concurrent with, and/or following spatially separating at least some ofthe plurality of capture objects into a plurality of locations. In somecases, the capture objects may be exposed to a plurality of bindingligands. In certain embodiments, a binding ligand may be adapted to bedirectly detected (e.g., the binding ligand comprises a detectablemolecule or moiety) or may be adapted to be indirectly detected (e.g.,including a component that can convert a precursor labeling agent into alabeling agent), as discussed more below. More than one type of bindingmay be employed in any given assay method, for example, a first type ofbinding ligand and a second type of binding ligand. In one example, thefirst type of binding ligand is able to associate with a first type ofanalyte molecule and the second type of binding ligand is able toassociate with the first binding ligand. In another example, both afirst type of binding ligand and a second type of binding ligand mayassociate with the same or different epitopes of a single analytemolecule, as described below.

Certain binding ligands can comprise a component that is able tofacilitate detection, either directly or indirectly. A component may beadapted to be directly detected in embodiments where the componentcomprises a measurable property (e.g., a fluorescence emission, a color,etc.). A component may facilitate indirect detection, for example, byconverting a precursor labeling agent into a labeling agent (e.g., anagent that is detected in an assay). A “precursor labeling agent” is anymolecule, particle, or the like, that can be converted to a labelingagent upon exposure to a suitable converting agent (e.g., an enzymaticcomponent). A “labeling agent” is any molecule, particle, or the like,that facilitates detection, by acting as the detected entity, using achosen detection technique.

In some embodiments, at least one binding ligand comprises an enzymaticcomponent. In some embodiments, the analyte molecule may comprise anenzymatic component. The enzymatic component may convert a precursorlabeling agent (e.g., an enzymatic substrate) into a labeling agent(e.g., a detectable product). A measure of the concentration of analytemolecules in the fluid sample can then be determined based at least inpart by determining the number of locations containing a labeling agent(e.g., by relating the number of locations containing a labeling agentto the number of locations containing an analyte molecule). Non-limitingexamples of enzymes or enzymatic components include horseradishperoxidase, beta-galactosidase, and alkaline phosphatase. Othernon-limiting examples of systems or methods for detection includeembodiments where nucleic acid precursors are replicated into multiplecopies or converted to a nucleic acid that can be detected readily, suchas the polymerase chain reaction (PCR), rolling circle amplification(RCA), ligation, Loop-Mediated Isothermal Amplification (LAMP), etc.Such systems and methods will be known to those of ordinary skill in theart, for example, as described in “DNA Amplification: CurrentTechnologies and Applications,” Vadim Demidov et al., 2004.

As an example of an assay method which comprises the use of a precursorlabeling agent, as shown in FIG. 3, substrate 100 comprising a pluralityof locations is provided, wherein the locations comprise reactionvessels. In reaction vessel 101 (e.g., location), analyte molecule 102is immobilized with respect to bead 103 (e.g., capture object). Bindingligand 104 is associated with analyte molecule 102. Binding ligand 104comprises an enzymatic component (not shown). Precursor labeling agent106 is converted to labeling agent 108 (upon exposure to the enzymaticcomponent). Labeling agent 108 is detected using methods describedherein. In contrast, reaction vessel 111 contains analyte molecule 112immobilized with respect to bead 110. In this reaction vessel, analytemolecule 112 is not associated with a binding ligand comprising anenzymatic component. Therefore, precursor labeling agent 114 is notconverted to a labeling agent in the reaction vessel. Thus this reactionvessel would give a different signal as compared to reaction vessel 101where the precursor labeling agent was converted to a labeling agent. Insome cases, there may also be reaction vessels which contain a bead notassociated with an analyte molecule, for example, reaction vessel 121contains bead 116. Additionally, some of the reaction vessels may notcomprise any bead, for example, reaction vessel 123. Reaction vessels121 and 123 may give different signals as compared to reaction vessel101 as there would be no labeling agent present. However, reactionvessels 121 and 123 may contain precursor labeling agent 117. More thanone precursor labeling agent may be present in any given reactionvessel.

In certain embodiments, solubilized, or suspended precursor labelingagents may be employed, wherein the precursor labeling agents areconverted to labeling agents which are insoluble in the liquid and/orwhich become immobilized within/near the location (e.g., within thereaction vessel in which the labeling agent is formed). Such precursorlabeling agents and labeling agents and their use is described incommonly owned U.S. patent application Ser. No. 12/236,484, entitled“High Sensitivity Determination of the Concentration of Analytemolecules in a Fluid Sample,” by Duffy, et al., filed Sep. 23, 2008,incorporated herein by reference.

In some embodiments, during the assay, at least one washing step may becarried out. In one instance, a plurality of capture objects may bewashed after exposing the capture objects to one or more solutionscomprising analyte molecules, binding ligands, precursor labelingagents, or the like. For example, following immobilization of theanalyte molecules with respect to a plurality of capture objects, theplurality of capture objects may be subjected to a washing step therebyremoving any analyte molecules not specifically immobilized with respectto a capture object. In certain embodiments, the wash solution isselected so that it does not cause appreciable change to theconfiguration of the capture objects and/or analyte molecules and/ordoes not disrupt any specific binding interaction between at least twocomponents of the assay (e.g., a capture component and an analytemolecule). In other cases, the wash solution may be a solution that isselected to chemically interact with one or more assay components. Aswill be understood by those of ordinary skill in the art, a wash stepmay be performed at any appropriate time point during the inventivemethods.

In some embodiments, assay methods may be carried out that do notcomprise the use of a plurality of capture objects comprising a bindingsurface for at least one type of analyte molecule and/or a plurality oflocations to which the capture objects may be spatially separated. Forexample, an assay according to the invention in certain embodiments mayuse any suitable method which is capable of isolating single analytemolecules and/or capture objects associated with one or more analytemolecules such that they can be individually addressed for detection.For example, an assay method may comprise providing a plurality ofcapture objects which are each associated with either a single analytemolecule or are free of any analyte molecules. At least a portion of thecapture objects may be individually addressed to determine the number ofthe capture objects associated with an analyte molecule or particle.Based at least in part on the number of capture objects determined to beassociated with an analyte molecule, a measure of the concentration ofanalyte molecules or particles in a fluid sample may be determined.

FIG. 4A illustrates a non-limiting embodiment where single analytemolecules are spatially segregating into a plurality of droplets. InFIG. 4A, plurality of analyte molecules 70 are provided, as shown instep (A). In this example, analyte molecules 70 are capable of beingoptically detected (e.g., the analyte molecules may be directly detectedusing optical interrogation). At least some of the plurality of analytemolecules 70 are contained within liquid droplets 72 (e.g., usingmicrofluidic techniques) which comprise fluid 71, as shown in step (B).Additionally, some droplets may be present which do not contain anyanalyte molecules (e.g., droplets 74 comprising fluid 71). Plurality ofdroplets 75 are substantially surrounded by fluid 73 which issubstantially immiscible with fluid 71. Plurality of droplets 75 can beoptically interrogated by feeding droplets into column 74 such that eachdroplet passes by an optical detection system (e.g., comprising lightsource 76 and detector 78) single file, as shown in step (C). Eachdroplet may be determined to contain an analyte molecule when there is achange in the optical single (e.g., a change in optical signal due tothe presence of an analyte molecule in the droplet).

As another example, as illustrated in FIG. 4B, plurality of analytemolecules 80 are provided, as shown in step (A). In this example,analyte molecules 70 are not capable of being optically detected, andmust be indirectly detection (as described herein). Plurality of analytemolecules 80 are exposed to a plurality of binding ligands 82, such thatat least one binding ligand associates with a significant portion of theanalyte molecules, as shown in step (B), to form complex 83, as shown instep (B). In this example, each binding ligand 82 comprises enzymaticcomponent 84. At least a portion of complexes 83 may be contained indroplets 85 (e.g., using microfluidic techniques), as shown in step (C),which comprise liquid 79. Additionally, some droplets may be presentwhich do not contain any complexes (e.g., droplets 86 comprising fluid79). Plurality of droplets 91 are substantially surrounded by fluid 87which is substantially immiscible with fluid 79. Droplets 85 and 86 mayadditionally comprise precursor labeling agent 86, which is converted tolabeling agent 88 upon exposure to enzymatic component 84, as indicatedby arrow 87. Plurality of droplets 91 can be optically interrogated byfeeding the plurality of droplets into column 89 such that each dropletpasses by an optical detection system (e.g., comprising light source 90and detector 92) single file, as shown in step (D). Each droplet may bedetermined to contain an analyte molecule when there is a change in theoptical single (e.g., a change in optical signal due to the presence ofa labeling agent in the droplet).

As yet another example, FIG. 4C illustrates and embodiment where singleanalyte molecules 282 are associated with respect to objects 280 viacapture components 274. Additionally, in this example, the immobilizedanalyte molecules are associated with binding ligand 284. The dropletscan be optically interrogated by feeding droplets into column 287 suchthat each droplet passed by the optical detection system (e.g.,comprising light source 286 and detector 288) single file. Each dropletmay be determined to contain a binding ligand when there is a change inthe optical single (e.g., a change in optical signal due to the presenceof a binding ligand in the droplet).

The following sections provide additional information regarding methodsteps, materials, and parameters that may be used to practice the assaymethods described above.

Capture Objects and Spatial Locations for Capture Object Segregation

In some embodiments, the method and systems of the present inventionutilize a plurality of capture objects that each includes a bindingsurface having affinity for at least one type of analyte molecule. Theplurality of capture objects may be configured to be able to bespatially segregated from each other, that is, the capture objects maybe provided in a form such that the capture objects are capable of beingspatially separated into a plurality of locations. For example, theplurality of capture objects may comprise a plurality of beads (whichcan be of any shape, e.g., sphere-like, disks, rings, cube-like, etc.),a dispersion or suspension of particulates (e.g., a plurality ofparticles in suspension in a fluid), nanotubes, or the like. In someembodiments, the plurality of capture objects is insoluble orsubstantially insoluble in the solvent(s) or solution(s) utilized in theassay. In some cases, the capture objects are solid or substantiallysolid (e.g., is essentially free of pores), however, in some cases, theplurality of capture objects may be porous or substantially porous,hollow, partially hollow, etc. The plurality of capture objects may benon-absorbent, substantially non-absorbent, substantially absorbent, orabsorbent. In some cases, the capture objects may comprise a magneticmaterial, which as described herein, may facilitate certain aspect ofthe assay (e.g., washing step). In some cases, a capture object surfacemay also comprise a protective or passivating layer that can reduce orminimize non-specific binding events (e.g., analyte molecules, bindingligands, etc.).

In some embodiments, the capture objects each include a binding surfacehaving affinity for at least one type of analyte molecule of interest.The portion of the capture object which comprises a binding surface maybe selected or configured based upon the physical shape/characteristicsand properties of the capture objects (e.g., size, shape), and theformat of the assay. In some embodiments, substantially all of the outersurfaces of the capture objects form the binding surfaces. A bindingsurface having an affinity for at least one type of analyte molecule maybe formed via the association of a plurality of capture components witha capture object. In some cases, an analyte molecule may associate witha capture component (e.g., become immobilized with respect to) viaformation of at least one chemical bond and/or physical adsorption, orcombination thereof. Non-limiting examples of types of chemical bondsinclude ionic bonds, covalent bonds (e.g., carbon-carbon, carbon-oxygen,oxygen-silicon, sulfur-sulfur, phosphorus-nitrogen, carbon-nitrogen,metal-oxygen, or other covalent bonds), hydrogen bonds (e.g., betweenhydroxyl, amine, carboxyl, thiol, and/or similar functional groups),dative bonds (e.g., complexation or chelation between metal ions andmonodentate or multidentate ligands), Van der Waals interactions, or thelike. Capture components which are useful or potentially useful forpracticing certain aspects and embodiments of the invention arediscussed in more detail below. At least some of the analyte molecules,upon exposure to the plurality of capture objects comprising a pluralityof capture components, become immobilized with respect to a capturecomponent. In certain embodiments, substantially all of the plurality ofanalyte molecules in the fluid sample tested may become immobilized withrespect to the capture components (and hence, the capture objects).

Without wishing to be bound by any theory, the use in certainembodiments of the invention of a capture step in which a plurality ofcapture objects having a large surface area for binding are exposed to afluid sample containing the analyte molecules or particles such that theanalyte molecules/particles become immobilized with respect to thecapture objects may facilitate an increase in the speed and/orefficiency of the assay for detection and quantification ofconcentration of the analyte in the sample compared to assays where theanalyte molecules themselves are segregated for detection without beingexposed to and immobilized with respect to a capture object. Thisincrease in binding speed and efficiency may be further enhanced if thesolution in which the plurality of analyte molecules and capture objectsare incubated for capture is agitated (e.g., stirred) to increase thecollision frequency and mass transfer rate between the plurality ofcapture objects (e.g., plurality of beads) and the analyte molecules(e.g., as contrasted with a substrate comprising a stationary surface(e.g., a microtiter plate)).

The plurality of capture objects for analyte capture may be of anysuitable size or shape. Non-limiting examples of suitable shapes includespheres, cubes, ellipsoids, tubes, sheets, and the like. In certainembodiments, the average diameter (if substantially spherical) oraverage maximum cross-sectional dimension (for other shapes) of acapture object may be greater than about 0.1 um (micrometer), greaterthan about 1 um, greater than about 10 um, greater than about 100 um,greater than about 1 mm, or the like. In other embodiments, the averagediameter of a capture object or the maximum dimension of a captureobject in one dimension may be between about 0.1 um and about 100 um,between about 1 um and about 100 um, between about 10 um and about 100um, between about 0.1 um and about 1 mm, between about 1 um and about 10mm, between about 0.1 um and about 10 um, or the like. The “averagediameter” or “average maximum cross-sectional dimension” of a pluralityof capture objects, as used herein, is the arithmetic average of thediameters/maximum cross-sectional dimensions of the capture objects.Those of ordinary skill in the art will be able to determine the averagediameter/maximum cross-sectional dimension of a population of captureobjects, for example, using laser light scattering, microscopy, sieveanalysis, or other known techniques. For example, in some cases, aCoulter counter may be used to determine the average diameter of aplurality of beads.

The capture objects for analyte capture may be fabricated from one ormore suitable materials, for example, plastics or synthetic polymers(e.g., polyethylene, polypropylene, polystyrene, polyamide,polyurethane, phenolic polymers, or nitrocellulose etc.), naturallyderived polymers (latex rubber, polysaccharides, polypeptides, etc),composite materials, ceramics, silica or silica-based materials, carbon,metals or metal compounds (e.g., comprising gold, silver, steel,aluminum, copper, etc.), inorganic glasses, silica, and a variety ofother suitable materials. Non-limiting examples of potentially suitableconfigurations include beads (e.g., magnetic beads), nanotubes, plates,disks, dipsticks, or the like.

In some embodiments, more than one type of capture object for analytecapture may be employed. In some cases, each type of capture object mayinclude a surface with differing binding specificity. In theseembodiments, more than one type of analyte molecule may be quantifiedand/or detected in a single, multiplexed assay method. For example, theplurality of capture objects for analyte capture may comprise aplurality of a first type of capture object comprising a binding surfacehaving an affinity for a first type of analyte molecule and a pluralityof a second type of capture objects comprising a binding surface havingan affinity for a second type of analyte molecule. Upon exposure to asample containing the first type of analyte molecule and the second typeof analyte molecule, the first type of analyte molecule becomesimmobilized with respect to the first type of capture object and thesecond type of analyte molecule becomes immobilized with respect to thesecond type of capture object. The first type of capture object and thesecond type of capture object may be encoded to be distinguishable fromeach other (e.g., to facilitate differentiation upon detection) byincluding a differing detectable property. For example, each type ofcapture object may have a differing fluorescence emission, a spectralreflectivity, shape, a spectral absorption, or an FTIR emission orabsorption. In a particular embodiment, each type of capture object maycomprise one or more dye compounds (e.g., fluorescent dyes) but atvarying concentration levels, such that each type of capture object hasa distinctive signal (e.g., based on the intensity of the fluorescentemission). Upon spatially segregating the capture objects after thecapture step into a plurality of locations for detection, a locationcomprising a first type of capture object associated with a first typeof analyte molecule may be distinguished from a location comprising asecond type of capture object associated with a second type of analytemolecule via detection of the differing property. The number oflocations comprising each type of capture object and/or the number ofcapture objects associated with an analyte molecule may be determined,enabling a determination of a measure of the concentration of both thefirst type of analyte molecule and the second type of analyte moleculesin the fluid sample based at least in part on these numbers.

For example, as illustrated in FIG. 5, step (A) a plurality of a firsttype of capture object 132 comprising a first type of capture component134 and a plurality of a second type of capture object 136 comprising asecond type of capture component 138 are provided. The plurality ofcapture objects is exposed to a fluid sample comprising a plurality of afirst type of analyte molecule 140 and a second type of analyte molecule142. As shown in step (B), at least some of the first type of analytemolecule 140 may associate with a capture object of the first type 132via capture component 134 and at least some of the second type ofanalyte molecule 142 may associate with a capture object of the secondtype 136 via capture component 138. Some of the first type of captureobjects and second type of capture objects may not associate with anyfirst type of or second type of analyte molecules. At least some of theplurality of capture objects from step (B) may be spatially segregatedinto a plurality of locations (represented by reaction vessels 142formed in substrate 146, as shown in step (C). Some of the reactionvessels may not comprise any capture objects. The plurality of reactionvessels may then be analyzed (e.g., for example, as illustrated in FIG.1, step (D)) to determine the number of reaction vessels containing afirst type of capture object associated with a first type of analytemolecule (e.g., reaction vessel 144) and the number of reaction vesselscontaining a second type of capture object associated with a second typeof analyte molecule (e.g., reaction vessel 145). Additionally, thenumber of locations containing a first type of capture object or asecond type of capture object not associated with any analyte moleculesmay also be determined. A measure of the concentration of the first type(or second type) of analyte molecule may be determined at least in partbased on the number of the first type (or second type) of analytemolecule detected. Alternatively, a measure of the concentration ofeither first type (or second type) of analyte molecule in the fluidsample may be based on the ratio of the number of reaction vesselscomprising the first type (or second type) of capture object associatedwith a first type (or second type) of analyte molecule to the number ofreactions vessels comprising the first type (or second type) of captureobjects not associated with any analyte molecules. Additional methodsfor determining the concentration of the first and/or second types ofanalyte molecules in the fluid sample may be carried out using methodssimilar to those described herein for samples comprising a single typeof analyte molecule. Using optical detection, the first type of captureobject may have a maximum wavelength of emission at a first wavelengthand the second type of capture object may maximum wavelength of emissionat a second wavelength, and therefore, allow for the reaction vesselswhich contain a first type of capture object to be distinguished fromthe reaction vessels which contain a second type of capture object.

Alternatively, or in combination, with use of coded capture objects formultiplexing, as described above, in some multiplexed assays, similarcapture object types may be employed that may, in certain cases, eachinclude capture components specific for multiple types of analytes. Incertain such assays, a first type of binding ligand, e.g., havingaffinity for a first analyte molecule type, and a second, third, etc.type of binding ligand having affinity for a second, third, etc.,respectively type of analyte molecule type and configured to bedetectably distinguishable from each other (e.g., through use ofdiffering detectable markers, enzymatic components and/or labelingagents, etc.) may be used in conjunction with the assay, and thedetection/quantification of the different types of binding ligands maybe correlated to the presence/concentration of different types ofanalyte molecules in the fluid sample.

In a particular embodiment, the plurality of capture objects for analytecapture comprises a plurality of beads. The beads may each comprise aplurality of capture components via which a plurality of analytemolecules may be immobilized. The plurality of capture components may bepresent on the surface of the beads. In some embodiments, the beads maybe magnetic beads. The magnetic property of the beads may help inseparating the beads from a solution (e.g., comprising a plurality ofunbound analyte molecules) and/or during washing step(s) (e.g., toremove excess fluid sample, labeling agents, etc.). Potentially suitablebeads, including magnetic beads, are available from a number ofcommercial suppliers. As noted above, there are many other examples ofpotentially suitable capture objects for analyte capture includingnanotubes (e.g., carbon nanotubes), microfluidic droplets (e.g.,droplets of a first fluid substantially surrounded by a second fluid),etc.

Those of ordinary skill in the art will be aware of methods andtechniques for exposing a plurality of capture objects to a fluid samplecontaining or suspected of containing an analyte molecule or particlefor initial analyte capture. For example, the plurality of captureobjects may be added (e.g., as a solid, as a solution) directly to afluid sample. As another example, the fluid sample may be added to theplurality of capture objects (e.g., in solution, as a solid). In someinstances, the solutions may be agitated (e.g., stirred, shaken, etc.).

Following immobilization of the analyte molecules with respect to aplurality of capture objects, the capture objects may be subjected to atleast one wash step. The wash step may aid in the removal of any unboundmolecules (e.g., analyte molecules, or other reaction components) fromthe solution. For example, referring to FIG. 1, following immobilizationof analyte molecules 4 with beads 6, as shown in step (B), a wash stepmay be performed to remove any unbound analyte molecules not immobilizedwith respect to an analyte molecule. As another example, referring toFIG. 2, following association of binding ligands 31 with analytemolecules 36, as shown in step (C), a wash step may be performed toremove any unbound binding ligands. The wash step may be performed usingany suitable technique known to those of ordinary skill in the art, forexample, by incubation of the capture objects with a wash solutionfollowed by centrifuging the solution comprising the capture objects anddecanting off the liquid, or by using filtration techniques. Inembodiments where the plurality of capture objects comprises a pluralityof magnetic beads, the beads may be isolated from the bulk solution withaid of a magnet.

The plurality of capture objects subsequent to the capture step (e.g.,at least some associated with at least one analyte molecule) may beexposed to one or more additional reagents, prior to spatiallysegregating the plurality of capture objects into a plurality oflocations for detection. For example, as noted previously, the captureobjects may be exposed a plurality of binding ligands, at least some ofwhich may associate with an immobilized analyte molecule. The captureobjects may be exposed to more than one type of binding ligand (e.g., afirst type of binding ligand and a second, third, etc. type of a bindingligand), as noted above. The association of a binding ligand with animmobilized analyte molecule may aid in the detection of the analytemolecules, as described herein.

In some embodiments, in addition to a plurality of capture objects foranalyte capture, a plurality of control objects may also be providedand/or employed. A control object(s) may be useful for a variety ofpurposes including, but not limited to, identification of theorientation of the plurality of locations (e.g., in the case where theplurality of locations is formed as an array of reaction sites, reactionvessels, etc.)—to help determine the quality of the assay, and/or tohelp calibrate the detection system (e.g., optical interrogationsystem), as described below. It should be understood, that more than onetype of control object may be present in any assay format (e.g., a firsttype of control object to determine quality of the assay and a secondtype of control object to act as a location marker), or a single type ofcontrol object may have more than one of the above-described functions.

In some cases, the control objects used to identify the orientation ofthe plurality of locations (e.g., reaction vessels, sites, etc.) on anarray (e.g., function as location marker(s) for an array). For example,a control object may be randomly or specifically distributed on anarray, and may provide one or more reference locations for determiningthe orientation/position of the array. Such a feature may be useful whencomparing multiple images of a portion of the array at different timeintervals. That is, the positions of control objects in the array may beused to register the images. In some cases, the control objects may beuse to provide reference locations in embodiments where a plurality ofimages of small overlapping regions are being combined to form a largerimage.

The presence of control objects in an assay may provide informationregarding the quality of the assay. For example, if a location is foundto contain a control object comprising an enzymatic component but nolabeling agent is present (e.g., the product of which would be presentupon exposure of a control object comprising an enzymatic component to aprecursor labeling agent), this gives an indication that some aspect ofthe assay may not be functioning properly. For example, the quality ofthe reagents may be compromised (e.g., concentration of precursorlabeling agent is too low, decomposition of the precursor labelingagent, etc.), and/or perhaps not all of the locations were exposed tothe precursor labeling agent.

In some embodiments, the control objects may be used to calibration thedetection system. For example, the control objects may output an opticalsignal which may be used to calibration an optical detection system. Insome embodiments, the control objects can be characterized and dopedwith a particular characteristic (e.g., fluorescence, color, absorbance,etc.) which can act as a quality control check for the detection systemperformance.

The control object may be provided with the plurality of capture objectsfor analyte capture prior to exposure to a fluid sample containinganalyte molecules, or may be added at another point in the assay (e.g.,following exposure to the plurality of analyte molecules and/or bindingligands, and/or prior to spatially segregating the plurality of captureobjects into a plurality of locations). The control objects may bedistinguishable from the capture objects using techniques known to thoseof ordinary skill in the art. For example, in some embodiments, thecontrol objects may comprise a unique property (e.g., are encoded) ascompare to the capture objects comprising a binding surface for theanalyte molecules. For example, the control object may have a differentfluorescence emission, a spectral reflectivity, shape, a spectralabsorption, or an FTIR emission or absorption, as compared to thecapture objects. The percentage of control objects to total number ofobjects (e.g., capture objects and control objects) in the assay may beabout 0.0001%, about 0.0005%, about 0.001%, about 0.005%, about 0.01%,about 0.05%, about 0.1%, about 0.5%, about 1%, about 5%, or the like.

In some embodiments, the control objects are configured as negativebinding controls and may be of a similar shape and size as the captureobjects used for immobilizing analyte molecules, however, the controlobjects may lack a binding surface for the analyte molecules (e.g., aplurality of capture components). For example, the control objects maycomprise a plurality of beads, and the capture objects for immobilizingthe analyte molecules may comprise the same or similar beads,additionally comprising at least one surface comprising a plurality ofcapture components.

In one embodiment, the control objects may comprise a positive controland include an enzymatic component. A precursor labeling agent may beconverted to a labeling agent upon exposure to the enzymatic component.In some cases, the enzymatic component may be the same as the enzymaticcomponent being used to detect the analyte molecules in a fluid sample(e.g., comprised in another component of the assay, for example, anenzymatic component comprised in a binding ligand, an analyte molecule,etc.). In such embodiments, the control object may be distinguishablefrom the capture objects such that the reaction vessels having apositive signal may be analyzed to determine whether the reaction vesselcomprises a control object (e.g., having a first detectable signal) or acapture object (e.g., having a second detectable signal distinguishablefrom the first detectable signal). In other cases, the enzymaticcomponent may be the different than an enzymatic component being used todetect the analyte molecules in a fluid sample (e.g., comprised inanother component of the assay, for example, an enzymatic componentcomprised in a binding ligand, an analyte molecule, etc.). In thisembodiment, the control object may or may not be distinguishable fromthe capture objects. Both a first type and a second type of precursorlabeling agent may be provided to the reaction vessels, and the firsttype of precursor labeling agent may be converted to a first type oflabeling agent upon exposure to the enzymatic component associated withthe control beads and the second type of precursor labeling agent may beconverted to a second type of labeling agent upon exposure to the otherenzymatic component (e.g., comprised in the binding ligand/analytemolecule/etc.). The reaction vessels containing the first type oflabeling agent correspond to the reaction vessels containing a controlobject and reaction vessels containing a second type of labeling agentcorrespond to the reaction vessels which contain a bindingligand/analyte molecule/etc. The plurality of locations containing acontrol bead may be observed to analyze, for example, the effectivenessof the enzymatic conversion reaction

A variety of methods may be used to prepare the controls objects, forexample, similar methods described herein for the capture objects (e.g.,beads comprising a plurality of capture components). In some cases, someof the control objects may comprise an enzymatic component. The controlobjects may be prepared such that 1) the majority of control objectseach comprise at least one enzymatic component (e.g., one, two, three,four, etc.) or 2) some of the control objects comprise a singleenzymatic component and the remainder of the control objects do notcomprise any enzymatic component. In the first case, during formation ofthe control objects, the ratio of enzymatic components provided insolution to objects may be greater than 1:1, greater than about 2:1,greater than about 5:1, greater than about 10:1, or the like. In suchcases, it would be expected that after partitioning the control objectson a substrate, each location comprising a control object would give apositive signal on exposure to a precursor labeling agent. In the secondcase, during formation of the control objects, the ratio of enzymaticcomponents provided in solution to objects may be less than about 1:5,less than about 1:10, less than about 1:20, less than about 1:50, or thelike. In such cases, it would be expected that ratio of the number oflocations comprising a control object and giving a positive signal tothe number of locations comprising a control object not giving apositive signal would be approximately similar to the ratio of enzymaticcomponents to objects during formation of the control objects and/or mayfollow a Poisson distribution.

As described above, following immobilization of a plurality of analytemolecules with respect to the plurality of capture objects in theanalyte capture step, at least a portion of the capture objects may bespatially segregated into a plurality of locations, for example on asubstrate. For example, each of capture objects of the portion ofcapture objects which are spatially segregated may be positioned inand/or associated with a location (e.g., a spot, region, well, etc. onthe surface and/or in the body of a substrate) that spatially distinctfrom the locations in which each of the other capture objects arelocated, such that the capture objects and locations can be individuallyresolved by an analytical detection system employed to address thelocations. As an example, each of a potion of the capture objects may bespatially segregated into an array of reaction vessels on a substrate,such that statistically only zero or one capture objects are located inat least some of the reaction vessels and in certain cases inessentially each reaction vessel. Each location may be individuallyaddressable relative to the other locations. Additionally, the locationsmay be arranged such that a plurality of locations may be addressedsubstantially simultaneously, as described herein, while stillpermitting the ability to resolve individual locations and captureobjects.

It should be understood, that while much of the discussion hereinfocusing on locations containing a single capture object, this is by nomeans limiting, and in some embodiments, more than one capture objectmay be contained at a single location. In such embodiments, the ratio ofcapture objects to analyte molecules may be such that following spatialsegregation of the plurality of capture objects into the plurality oflocations, a statistically significant fraction of the locations containno analyte molecules and a statistically significant fraction oflocations contain at least one analyte molecule. That is, while a singlelocation may contain a plurality of capture objects, in some cases, noneof the capture objects are associated with any analyte molecules andonly a single one of the capture objects in an addressed location isassociated with at least one analyte molecule.

As noted above, in some embodiments, the plurality of locationscomprises a plurality of reaction vessels/wells on a substrate. Thereactions vessels, in certain embodiments, may be configured to receiveand contain only a single capture object used for analyte capture. Theplurality of capture objects can be partitioned across a plurality ofsuch reaction vessels (e.g., configured as an array of reaction vesselson a substrate), in some cases, to facilitate determination of a measureof the concentration of analyte molecules in a fluid sample by meansdiscussed in further detail below and in the examples.

In some embodiments of the present invention, the plurality of reactionvessels may be sealed e.g., after the introduction of the captureobjects used for analyte capture, for example, through the mating of thesecond substrate and a sealing component. The sealing of the reactionvessels may be such that the contents of each reaction vessel cannotescape the reaction vessel during the remainder of the assay. In somecases, the reaction vessels may be sealed after the addition of thecapture objects and, optionally, a precursor labeling agent tofacilitate detection of the analyte molecules. For embodiments employingprecursor labeling agents, by sealing the contents in some or eachreaction vessel, a reaction to produce the detectable labeling agentscan proceed within the sealed reaction vessels, thereby producing adetectable amount of labeling agents that is retained in the reactionvessel for detection purposes.

The plurality of locations comprising a plurality of reaction vesselsmay be formed using a variety of methods and/or materials. In somecases, the plurality of reaction vessels is formed as an array ofdepressions on a first surface. In other cases, however, the pluralityof reaction vessels may be formed by mating a sealing componentcomprising a plurality of depressions with a substrate that may eitherhave a featureless surface or include depressions aligned with those onthe sealing component. Any of the device components, for example, thesubstrate or sealing component, may be fabricated from a compliantmaterial, e.g., an elastomeric polymer material, to aid in sealing. Thesurfaces may be or made to be hydrophobic or contain hydrophobic regionsto minimize leakage of aqueous samples from the microwells.

In some cases, the sealing component may be capable of contacting theexterior surface of an array of microwells (e.g., the cladding of afiber optic bundle as described in more detail below) such that eachreaction vessel becomes sealed or isolated such that the contents ofeach reaction vessel cannot escape the reaction vessel. According to oneembodiment, the sealing component may be a silicone elastomer gasketthat may be placed against an array of microwells with application ofsubstantially uniform pressure across the entire substrate. In somecases, the reaction vessels may be sealed after the addition of theplurality of capture objects used for analyte capture and, optionally,any precursor labeling agent molecule that may be used to facilitatedetection of the analyte molecule.

A non-limiting example of the formation of a plurality of reactionvessels containing assay solution on/in a substrate is depicted in FIG.6. FIG. 6, panel (A) shows a surface comprising a plurality ofmicrowells 139, which have been exposed to an assay solution 141 (e.g.,a solution containing the capture objects used for analyte captureand/or control objects obtained after performance of the analyte capturestep(s) and any washing step(s)), and a sealing component 143. Sealingcomponent 143 in this example comprises a substantially planar bottomsurface. Mating of substrate 139 with sealing component 143 forms aplurality of sealed reaction vessels 145. The areas between the reactionvessels 148 may be modified to aid in the formation of a tight sealbetween the reaction vessels.

A second embodiment is shown in FIG. 6, panel (B), in which sealingcomponent 162 comprising a plurality of microwells 163 is mated with asubstantially planar surface 158 which has been exposed to assaysolution 162, thereby forming a plurality of reaction vessels 164.

In a third embodiment, as shown in FIG. 6, panel (C), substrate surface166 comprising a plurality of microwells 167 is mated with sealingcomponent 170 also comprising a plurality of microwells 171. In thisembodiment, the microwells in the substrate and the microwells in thesealing components are substantially aligned so each reaction vessel 172formed comprises a portion of the microwell from the sealing componentand a portion of a microwell from the substrate. In FIG. 6, panel (D),the microwells are not aligned such that each reaction vessel compriseseither a microwell from the sealing component 173 or a microwell fromthe substrate 175.

The sealing component may be essentially the same size as the substrateor may be different in size. In some cases, the sealing component isapproximately the same size as the substrate and mates withsubstantially the entire surface of the substrate. In other cases, asdepicted in FIG. 6, panel (E), the sealing component 176 is smaller thanthe substrate 174 and the sealing component only mates with a portion178 of the substrate. In yet another embodiment, as depicted in FIG. 6,panel (F), the sealing component 182 is larger than the substrate 180,and only a portion 184 of the sealing component mates with the substrate180.

In some embodiments, the reaction vessels may all have approximately thesame volume. In other embodiments, the reaction vessels may havediffering volumes. The volume of each individual reaction vessel may beselected to be appropriate to facilitate any particular assay protocol.For example, in one set of embodiments where it is desirable to limitthe number of capture objects used for analyte capture contained in eachvessel to a small number, the volume of the reaction vessels may rangefrom attoliters or smaller to nanoliters or larger depending upon thenature of the capture objects, the detection technique and equipmentemployed, the number and density of the wells on the substrate and theexpected concentration of capture objects in the fluid applied to thesubstrate containing the wells. In one embodiment, the size of thereaction vessel may be selected such only a single capture object usedfor analyte capture can be fully contained within the reaction vessel.In accordance with one embodiment of the present invention, the reactionvessels may have a volume between about 1 femtoliter and about 1picoliter, between about 1 femtoliters and about 100 femtoliters,between about 10 attoliters and about 100 picoliters, between about 1picoliter and about 100 picoliters, between about 1 femtoliter and about1 picoliter, or between about 30 femtoliters and about 60 femtoliters.In some cases, the reaction vessels have a volume of less than about 1picoliter, less than about 500 femtoliters, less than about 100femtoliters, less than about 50 femtoliters, or less than about 1femtoliter. In some cases, the reaction vessels have a volume of about10 femtoliters, about 20 femtoliters, about 30 femtoliters, about 40femtoliters, about 50 femtoliters, about 60 femtoliters, about 70femtoliters, about 80 femtoliters, about 90 femtoliters, or about 100femtoliters.

In embodiments where the plurality of capture objects used for analytecapture comprise a plurality of beads and the plurality of locationscomprise a plurality of reaction vessels having a shape that isessentially that of a circular cylinder, the size of the reactionvessels may be based upon the size of the beads and may be designed soas to ensure that the number of wells containing more than a single beadis minimal. In some cases, the maximum permissible well diameter may becalculated according to Equation 3:

$\begin{matrix}{{2*{BeadRadius}} + \sqrt{\left( {{3*{BeadRadius}^{2}} - {WellDepth}^{2} + {2*{WellDepth}*{BeadRadius}}} \right)}} & \left( {{Eq}.\mspace{14mu} 3} \right)\end{matrix}$

and/or the maximum permissible well depth may be calculated according toEquation 4:

$\begin{matrix}{{BeadRadius} + \sqrt{\left( {{4*{BeadRadius}*{WellDiameter}} - {WellDiameter}^{2}} \right)}} & \left( {{Eq}.\mspace{14mu} 4} \right)\end{matrix}$

The minimum permissible well depth and the minimum permissible welldiameter to assure that a single bead can be contained in the well, inmost embodiments, will not be less than the average diameter of thebead. Having a properly sized reaction vessel which allows for no morethan a single bead to be present in a reaction vessel may provide betterability to resolve individual beads allowing for more accuracy withregard to determining a measure of the concentration of analytemolecules in a fluid sample by the means described in more detail belowand in the Examples. For example, if the reaction vessels are too large,more than one bead may be able to fit in the reaction vessel, which maylead to an increase in the number of reaction vessels containingmultiple analyte molecules, which may introduce inaccuracy in aconcentration determination using an algorithm/statistical model basedon single molecule detection (see below). In some cases, however, it maybe desirable to have more than one bead fit in a reaction vessel. On theother hand, in the reaction vessel is too small, a bead may not be ableto fit in the reaction vessel, thereby preventing proper sealing of thereaction vessel (e.g., in embodiments where the reaction vessel issealed) and/or may lead to difficulties in addressing individuallocations (e.g., in embodiments where a labeling agent is produced fordetection, the labeling agent may disperse away from the reaction vesselit is produced in). In such cases, there may be false positives (e.g., areaction vessel which does not contain an analyte molecule may bedetermined to contain an analyte molecule based on the labeling agentwhich has diffused away from the location in which it was produced)which may lead to imprecise determination of a measure of theconcentration of analyte molecules in a fluid sample.

In some embodiments, the average depth of the reaction vessels isbetween about 1.0 and about 1.7 times, between about 1.0 times and about1.5 times, between about 1.0 times and about 1.3 times, or between about1.1 times and about 1.4 times the average diameter of the beads. In someembodiments, the average diameter of the reactions vessels is betweenabout 1.0 times and about 1.9 times, between about 1.2 times and about1.7 times, between about 1.0 times and about 1.5 times, or between about1.3 times and about 1.6 times the average diameter of the beads. In aparticular embodiment, the average depth of the reaction vessels isbetween about 1.0 times and about 1.5 times the average diameter of thebeads and the average diameter of the reactions vessels is between about1.0 times and about 1.9 times the average diameter of the beads.

The total number of locations and/or density of the locations employedin an assay (e.g., the number/density of reaction vessels in an array)can depend on the composition and end use of the array. For example, thenumber of reaction vessels employed may depend on the number of captureobjects employed, the suspected concentration range of the assay, themethod of detection, the size of the capture objects, the type ofdetection entity (e.g., free labeling agent in solution, precipitatinglabeling agent, etc.). Arrays containing from about 2 to many billionsof reaction vessels (or total number of reaction vessels) can be made byutilizing a variety of techniques and materials. Increasing the numberof reaction vessels in the array can be used to increase the dynamicrange of an assay or to allow multiple samples or multiple types ofanalyte molecules to be assayed in parallel. The array may comprisebetween one thousand and one million reaction vessels per sample to beanalyzed. In some cases, the array comprises greater than one millionreaction vessels. In some embodiments, the array comprises between about1,000 and about 50,000, between about 1,000 and about 1,000,000, betweenabout 1,000 and about 10,000, between about 10,000 and about 100,000,between about 100,000 and about 1,000,000, between about 100,000 andabout 500,000, between about 1,000 and about 100,000, between about50,000 and about 100,000, between about 20,000 and about 80,000, betweenabout 30,000 and about 70,000, between about 40,000 and about 60,000, orthe like, reaction vessels. In some embodiments, the array comprisesabout 10,000, about 20,000, about 50,000, about 100,000, about 150,000,about 200,000, about 300,000, about 500,000, about 1,000,000, or more,reaction vessels.

The array of reaction vessels may be arranged on a substantially planarsurface or in a non-planar three-dimensional arrangement. The reactionvessels may be arrayed in a regular pattern or may be randomlydistributed. In a specific embodiment, the array is a regular pattern ofsites on a substantially planar surface permitting the sites to beaddressed in the X-Y coordinate plane.

In some embodiments, the reaction vessels are formed in a solidmaterial. As will be appreciated by those in the art, the number ofpotentially suitable materials in which the reaction vessels can beformed is very large, and includes, but is not limited to, glass(including modified and/or functionalized glass), plastics (includingacrylics, polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, cyclic olefincopolymer (COC), cyclic olefin polymer (COP), Teflon®, polysaccharides,nylon or nitrocellulose, etc.), elastomers (such as poly(dimethylsiloxane) and poly urethanes), composite materials, ceramics, silica orsilica-based materials (including silicon and modified silicon), carbon,metals, optical fiber bundles, or the like. In general, the substratematerial may be selected to allow for optical detection withoutappreciable autofluorescence. In certain embodiments, the reactionvessels may be formed in a flexible material.

A reaction vessel in a surface (e.g., substrate or sealing component)may be formed using a variety of techniques known in the art, including,but not limited to, photolithography, stamping techniques, moldingtechniques, etching techniques, or the like. As will be appreciated bythose of the ordinary skill in the art, the technique used can depend onthe composition and shape of the supporting material and the size andnumber of reaction vessels.

In a particular embodiment, an array of reaction vessels is formed bycreating microwells on one end of a fiber optic bundle and utilizing aplanar compliant surface as a sealing component. In certain suchembodiments, an array of reaction vessels in the end of a fiber opticbundle may be formed as follows. First, an array of microwells is etchedinto the end of a polished fiber optic bundle. Techniques and materialsfor forming and etching a fiber optic bundle are known to those ofordinary skill in the art. For example, the diameter of the opticalfibers, the presence, size and composition of core and cladding regionsof the fiber, and the depth and specificity of the etch may be varied bythe etching technique chosen so that microwells of the desired volumemay be formed. In certain embodiments, the etching process createsmicrowells by preferentially etching the core material of the individualglass fibers in the bundle such that each well is approximately alignedwith a single fiber and isolated from adjacent wells by the claddingmaterial. Potential advantages of the fiber optic array format is thatit can produce thousands to millions of reaction vessels withoutcomplicated microfabrication procedures and that it can provide theability to observe and optically address many reaction vesselssimultaneously.

Each microwell may be aligned with an optical fiber in the bundle sothat the fiber optic bundle can carry both excitation and emission lightto and from the wells, enabling remote interrogation of the wellcontents. Further, an array of optical fibers may provide the capabilityfor simultaneous or non-simultaneous excitation of molecules in adjacentvessels, without signal “cross-talk” between fibers. That is, excitationlight transmitted in one fiber does not escape to a neighboring fiber.

Alternatively, the equivalent structures of a plurality of reactionvessels may be fabricated using other methods and materials that do notutilize the ends of an optical fiber bundle as a substrate. For example,the array may be a spotted, printed or photolithographically fabricatedsubstrate produced by techniques known in the art; see for exampleWO95/25116; WO95/35505; PCT US98/09163; U.S. Pat. Nos. 5,700,637,5,807,522, 5,445,934, 6,406,845, and 6,482,593. In some cases, the arraymay be produced using molding, embossing, and/or etching techniques aswill be known to those of ordinary skill in the art.

In certain embodiments, the present invention provides a system equippedwith a mechanical platform that applies a sealing component to asubstrate. The platform may be positioned beneath a stage on the system.After the chosen reaction components have been added to an array ofreaction vessels, the sealing component may be mated with the array. Forexample, the sealing component may be sandwiched between a flat surface(such as, for example, a microscope slide) and the array of reactionvessels using uniform pressure applied by the mechanical platform.

A non-limiting embodiment is illustrated in FIG. 7. A sealing component300 is placed on top of mechanical platform 302. The assay solution 304is placed on top of the sealing component 300. The mechanical platformis moved upwards towards the array 306 (e.g., fiber optic array) suchthat uniform pressure is applied. As shown in FIG. 8, the sealingcomponent 300 forms a tight seal with the array 306. In other instances,varying pressure may be applied to the sealing component to form a tightseal between the sealing component and the array. The system may alsocomprise additional components 312 that may be utilized to analyze thearray (e.g., microscope, computer, etc.) as discussed more herein.

The plurality of capture objects used for analyte capture may bespatially separated into the plurality of reaction vessels using any ofa wide variety of techniques known to those of ordinary skill in theart. In some cases, the plurality of reaction vessels may be exposed toa solution containing the plurality of capture objects. In some cases,force may be applied to the solution and/or capture objects, therebyaiding in the spatial separation of the capture objects from the fluidphase and/or the deposition of the capture objects in the vessels. Forexample, after application of an assay solution containing the captureobjects to a substrate containing the reaction vessels, the substrateand solution may be centrifuged to assist in depositing the captureobjects in the reaction vessels. In embodiments where the captureobjects (e.g., beads) are magnetic, a magnet may be used to aid incontaining the capture objects in the reaction vessels. In some cases,when the plurality of reaction vessels is formed on the end of a fiberoptic bundle (or another planar surface), a material (e.g., tubing) maybe placed around the edges of the surface of the array comprising theplurality of reaction vessel to form a container to hold the solution inplace while the capture objects settle in the reaction vessels or areplaced into the reaction vessels (e.g., while centrifuging). Followingplacement of the capture objects into at least some of the reactionvessels, the surrounding material may be removed and the surface of thearray may be washed and/or swabbed to remove any excess solution/captureobjects.

In some embodiments, the substrate does not include wells or reactionvessels forming the plurality of reaction vessels but uses/providesother means to spatially segregate the plurality of capture objects usedfor analyte capture. In some cases, a patterned substantially planarsurface may be employed, wherein the patterned areas form a plurality oflocations. In some cases, the patterned areas may comprise substantiallyhydrophilic surfaces which are substantially surrounded by substantiallyhydrophobic surfaces. A plurality of capture objects (e.g., beads) maybe substantially surround by a substantially hydrophilic medium (e.g.,comprising water), and the beads may be exposed to the pattern surfacesuch that the beads associate in the patterned areas (e.g., thelocations), thereby spatially segregating the plurality of beads. Forexample, in one such embodiment, a substrate may be or include a gel orother material able to provide a sufficient barrier to mass transport(e.g., convective and/or diffusional barrier) to prevent capture objectsused for analyte capture and/or precursor labeling agent and/or labelingagent from moving from one location on or in the material to anotherlocation so as to cause interference or cross-talk between spatiallocations containing different capture objects during the time framerequired to address the locations and complete the assay. For example,in one embodiment, a plurality of capture objects is spatially separatedby dispersing the capture objects on and/or in a hydrogel material. Insome cases, a precursor labeling agent may be already present in thehydrogel, thereby facilitating development of a local concentration ofthe labeling agent (e.g., upon exposure to a binding ligand or analytemolecule carrying an enzymatic component). As still yet anotherembodiment, the capture objects may be confined in one or morecapillaries. In some cases, the plurality of capture objects may beabsorbed or localized on a porous or fibrous substrate, for example,filter paper. In some embodiments, the capture objects may be spatiallysegregated on a uniform surface (e.g., a planar surface), and thecapture objects may be detected using precursor labeling agents whichare converted to substantially insoluble or precipitating labelingagents that remain localized at or near the location of where thecorresponding capture object is localized. The use of such substantiallyinsoluble or precipitating labeling agents is described herein.

Articles and Kits

In some embodiments of the present invention, an article or kit fordetermining a measure of the concentration of analyte molecules orparticles in a fluid sample is provided. The article or kit may comprisea plurality of beads and a substrate comprising a plurality of reactionvessels. The reaction vessels may be configured to receive and containthe capture objects. The plurality of beads in certain embodiment havean average diameter between about 0.1 micrometer and about 100micrometers and the size of the reaction vessels may be selected suchthat only either zero or one beads is able to be contained in singlereaction vessels. In some cases, the average depth of the reactionvessels is between about 1.0 times and about 1.5 times the averagediameter of the beads and the average diameter of the reactions vesselsis between about 1.0 times and about 1.9 times the average diameter ofthe beads. In certain embodiments, the beads may have an averagediameter between about between about 1 micrometer and about 10micrometers, between about 1 micrometer and about 5 micrometers, or anyrange of sizes described herein.

The plurality of beads provided may have a variety of properties andparameters, as described herein. For example, the beads may be magnetic.The plurality of beads may comprise a binding surface (e.g., a pluralityof capture components) having an affinity for at least one type ofanalyte molecule or particle.

The plurality of reaction vessels may be formed in any suitablesubstrate, as described herein. In a particular embodiment, theplurality of reaction vessels is formed on the end of a fiber opticbundle. The fiber optic bundle may be prepared (e.g., etched) accordingto methods known to those of ordinary skill in the art and/or methodsdescribed herein. In other embodiments, the plurality of reactionsvessels is formed in a plate or similar substantially planar material(e.g., using lithography or other known techniques). Exemplary suitablematerials are described herein.

The average depth of the plurality of reaction vessels may be betweenabout 1.0 and about 1.5 times, or between about 1.1 and about 1.3 timesthe average diameter of the beads, or any range described herein. Theaverage diameter of the plurality of reaction vessels may be betweenabout 1.0 times and about 1.9 times, or between about 1.3 times andabout 1.8 times the average diameter of the beads, or any rangedescribed herein. The average depth and/or the average diameter of theplurality of reaction vessels may be chosen such that no more than onebead is able to be contained in a reaction vessels. Methods forcalculating maximum depths and maximum diameters to facilitate singlebead loading are described herein. The average volume of the pluralityof reaction vessels may be between about 10 attoliters and about 100picoliters, between about 1 femtoliter and about 1 picoliter, or anydesired range. The substrate may comprise any number of reactionvessels, for example, between about 1,000 and about 1,000,000 reactionvessels, between about 10,000 and about 100,000 reaction vessels, orbetween about 100,000 and about 300,000 reaction vessels, or any otherdesired range.

The article or kit may comprise any number of additional components,some of which are described in detail herein. In some cases, the articleor kit may further comprise a sealing component configured for sealingthe plurality of reaction vessels. In certain embodiments, the pluralityof reaction vessels may be formed upon the mating of at least a portionof a sealing component and at least a portion of the second substrate,as shown in FIGS. 7A-7F and as discussed in more detail herein. Asanother example, the kit may also provide solutions for carrying out anassay method as described herein. Non-limiting example of solutionsinclude solutions containing one or more types of binding ligands andprecursor labeling agents. In some cases, the article or kit maycomprise at least one type of control bead.

In some embodiments, the kit may optionally include instructions for useof the plurality of beads and the plurality or reaction vessels (and anyadditional components provided). That is, the kit can include adescription of use of the beads and reaction vessels, for example, foruse with a system to determine a measure of the concentration of analytemolecules (or particles) in a fluid sample. As used herein,“instructions” can define a component of instruction and/or promotion,and typically involve written instructions on or associated withpackaging of the invention. Instructions also can include any oral orelectronic instructions provided in any manner such that a user of thekit will clearly recognize that the instructions are to be associatedwith the kit. Additionally, the kit may include other componentsdepending on the specific application, as described herein. As usedherein, “promoted” includes all methods of doing business includingmethods of education, hospital and other clinical instruction,scientific inquiry, drug discovery or development, academic research,pharmaceutical industry activity including pharmaceutical sales, and anyadvertising or other promotional activity including written, oral andelectronic communication of any form, associated with the invention.

Capture Components

In some embodiments of the present invention, the surface of the captureobjects may provide a binding surface having an affinity for at leastone type of analyte molecule or particle. In some embodiments, thebinding surface may comprise at least one type of capture component.Generally, the capture component allows the attachment of a molecule,particle, or complex to a solid support (that is, a surface of a captureobject) for the purposes of immobilization, detection, quantification,and/or other analysis of the molecule, particle, or complex. A capturecomponent is used in the present invention, in some cases, to immobilizean analyte molecule with respect to a capture object (e.g., a bead).

As will be appreciated by those in the art, the composition of thecapture component will depend on the composition of the analytemolecule. Capture components for a wide variety of target molecules areknown or can be readily found or developed using known techniques. Forexample, when the target molecule is a protein, the capture componentsmay comprise proteins, particularly antibodies or fragments thereof(e.g., antigen-binding fragments (Fabs), Fab′ fragments, pepsinfragments, F(ab′)₂ fragments, full-length polyclonal or monoclonalantibodies, antibody-like fragments, etc.), other proteins, such asreceptor proteins, Protein A, Protein C, etc., or small molecules. Insome cases, capture components for proteins comprise peptides. Forexample, when the target molecule is an enzyme, suitable capturecomponents may include enzyme substrates and/or enzyme inhibitors. Insome cases, when the target analyte is a phosphorylated species, thecapture component may comprise a phosphate-binding agent. For example,the phosphate-binding agent may comprise metal-ion affinity media suchas those describe in U.S. Pat. No. 7,070,921 and U.S. Patent ApplicationNo. 20060121544. In addition, when the target molecule is asingle-stranded nucleic acid, the capture component may be acomplementary nucleic acid. Similarly, the target molecule may be anucleic acid binding protein and the capture component may be asingle-stranded or double-stranded nucleic acid; alternatively, thecapture component may be a nucleic acid-binding protein when the targetmolecule is a single or double stranded nucleic acid. Alternatively, asis generally described in U.S. Pat. Nos. 5,270,163, 5,475,096,5,567,588, 5,595,877, 5,637,459, 5,683,867, 5,705,337, and relatedpatents, nucleic acid “aptamers” may be developed for capturingvirtually any target molecule. Also, for example, when the targetmolecule is a carbohydrate, potentially suitable capture componentsinclude, for example, antibodies, lectins, and selectins. As will beappreciated by those of ordinary skill in the art, any molecule that canspecifically associate with a target molecule of interest maypotentially be used as a capture component.

For certain embodiments, suitable target analyte molecule/capturecomponent pairs can include, but are not limited to,antibodies/antigens, receptors/ligands, proteins/nucleic acid, nucleicacids/nucleic acids, enzymes/substrates and/or inhibitors, carbohydrates(including glycoproteins and glycolipids)/lectins and/or selectins,proteins/proteins, proteins/small molecules; small molecules/smallmolecules, etc. According to one embodiment, the capture components areportions (particularly the extracellular portions) of cell surfacereceptors that are known to multimerize, such as the growth hormonereceptor, glucose transporters (particularly GLUT 4 receptor), andT-cell receptors and the target analyte molecules are one or morereceptor target ligands.

In a particular embodiment, the capture component may be attached to thesurface of a capture object via a linkage, which may comprise anymoiety, functionalization, or modification of the binding surface and/orcapture component that facilitates the attachment of the capturecomponent to the surface. The linkage between the capture component andthe surface may comprise one or more chemical or physical (e.g.,non-specific attachment via van der Waals forces, hydrogen bonding,electrostatic interactions, hydrophobic/hydrophilic interactions; etc.)bonds and/or chemical linkers providing such bond(s). In certainembodiments, the capture component comprises a capture extendercomponent. In such embodiments, the capture component comprises a firstportion that binds the analyte molecule and a second portion that can beused for attachment to the binding surface.

In certain embodiments, a capture object surface may also comprise aprotective or passivating layer that can reduce or minimize non-specificattachment of non-capture components (e.g., analyte molecules, bindingligands) to the binding surface during the assay which may lead to falsepositive signals during detection or to loss of signal. Examples ofmaterials that may be utilized in certain embodiments to formpassivating layers include, but are not limited to: polymers, such aspoly(ethylene glycol), that repel the non-specific binding of proteins;naturally occurring proteins with this property, such as serum albuminand casein; surfactants, e.g., zwitterionic surfactants, such assulfobetaines; naturally occurring long-chain lipids; and nucleic acids,such as salmon sperm DNA.

The method of attachment of the capture component to a capture objectsurface depends of the type of linkage employed and may potentially beaccomplished by a wide variety of suitable couplingchemistries/techniques known to those of ordinary skill in the art. Theparticular means of attachment selected will depend on the materialcharacteristics of the capture object surface and the nature of thecapture component. In certain embodiments, the capture components may beattached to the capture object surface through the use of reactivefunctional groups on each. According to one embodiment, the functionalgroups are chemical functionalities. That is, the binding surface may bederivatized such that a chemical functionality is presented at thebinding surface which can react with a chemical functionality on thecapture component resulting in attachment. Examples of functional groupsfor attachment that may be useful include, but are not limited to, aminogroups, carboxy groups, epoxide groups, maleimide groups, oxo groups,and thiol groups. Functional groups can be attached, either directly orthrough the use of a linker, the combination of which is sometimesreferred to herein as a “crosslinker.” Crosslinkers are known in theart; for example, homo- or hetero-bifunctional crosslinkers as are wellknown (e.g., see 1994 Pierce Chemical Company catalog, technical sectionon crosslinkers, pages 155-200, or “Bioconjugate Techniques” by Greg T.Hermanson, Academic Press, 1996). Non-limiting example of crosslinkersinclude alkyl groups (including substituted alkyl groups and alkylgroups containing heteroatom moieties), esters, amide, amine, epoxygroups and ethylene glycol and derivatives. A crosslinker may alsocomprise a sulfone group, forming a sulfonamide.

According to one embodiment, the functional group is a light-activatedfunctional group. That is, the functional group can be activated bylight to attach the capture component to the capture object surface. Oneexample is PhotoLink™ technology available from SurModics, Inc. in EdenPrairie, Minn.

In some cases, the capture object may comprise streptavidin-coatedsurfaces and the capture component may be biotinylated. Exposure of thecapture component to the streptavidin-coated surfaces can causeassociation of the capture component with the surface by interactionbetween the biotin component and streptavidin.

In certain embodiments, attachment of the capture component to thebinding surface may be effected without covalently modifying the bindingsurface of a capture object. For example, the attachment functionalitycan be added to the binding surface by using a linker that has both afunctional group reactive with the capture component and a group thathas binding affinity for the binding surface. In certain embodiments, alinker comprises a protein capable of binding or sticking to the bindingsurface; for example, in one such embodiment, the linker is serumalbumin with free amine groups on its surface. A second linker(crosslinker) can then be added to attach the amine groups of thealbumin to the capture component (e.g., to carboxy groups).

According to one embodiment in which a chemical crosslinker is used toattach the capture components to the capture object, the analytemolecule may be captured on the binding surface of a capture objectusing a capture component attached via chemical crosslinking in thefollowing manner. First, the binding surface is derivatized with afunctional group, such as, an amine group. Next, a crosslinker and thecapture component are placed in contact with the binding surface suchthat one end of the crosslinker attaches to the amine group and thecapture component attaches to the other end of the crosslinker. In thisway, capture components comprising proteins, lectins, nucleic acids,small organic molecules, carbohydrates can be attached.

One embodiment utilizes proteinaceous capture components. As is known inthe art, any number of techniques may be used to attach a proteinaceouscapture component to a wide variety of solid surfaces. “Protein” or“proteinaceous” in this context includes proteins, polypeptides,peptides, including, for example, enzymes, and antibodies. A widevariety of techniques are known to add reactive moieties to proteins,for example, the method outlined in U.S. Pat. No. 5,620,850. Theattachment of proteins to surfaces is known, for example, see Heller,Acc. Chem. Res. 23:128 (1990), and many other similar references.

In some embodiments, the capture component (or binding ligand) maycomprise Fab′ fragments. The use of Fab′ fragments as opposed to wholeantibodies may help reduce non-specific binding between the capturecomponent and the binding ligand. In some cases, the Fc region of acapture component (or binding ligand) may be removed (e.g.,proteolytically). In some cases, an enzyme may be used to remove the Fcregion (e.g., pepsin, which may produce F(ab′)₂ fragments and papain,which may produce Fab fragments). In some instances, the capturecomponent may be attached to a binding surface using amines or may bemodified with biotin (e.g., using NHS-biotin) to facilitate binding toan avidin or streptavidin coated capture object surface. F(ab′)₂fragments may be subjected to a chemical reduction treatment (e.g., byexposure to 2-mercaptoethylamine) to, in some cases, form twothiol-bearing Fab′ fragments. These thiol-bearing fragments can then beattached via reaction with a Michael acceptor such as maleimide. Forexample, the Fab′ fragments may then be treated with a reagent (e.g.,maleimide-biotin) to attach at least one biotin moiety (i.e.,biotinylated) to facilitate attachment to streptavidin-coated surfacesas described above.

Certain embodiments utilize nucleic acids as the capture component, forexample for when the analyte molecule is a nucleic acid or a nucleicacid binding protein, or when the it is desired that the capturecomponent serve as an aptamer for binding a protein, as is well known inthe art.

According to one embodiment, each binding surface of a capture objectcomprises a plurality of capture components. The plurality of capturecomponents, in some cases, may be distributed randomly on the bindingsurface like a “lawn.” Alternatively, the capture components may bespatially segregated into distinct region(s) and distributed in anydesired fashion.

Binding between the capture component and the analyte molecule, incertain embodiments, is specific, e.g., as when the capture componentand the analyte molecule are complementary parts of a binding pair. Incertain such embodiments, the capture component binds both specificallyand directly to the analyte molecule. By “specifically bind” or “bindingspecificity,” it is meant that the capture component binds the analytemolecule with specificity sufficient to differentiate between theanalyte molecule and other components or contaminants of the testsample. For example, the capture component, according to one embodiment,may be an antibody that binds specifically to some portion of an analytemolecule (e.g., an antigen). The antibody, according to one embodiment,can be any antibody capable of binding specifically to an analytemolecule of interest. For example, appropriate antibodies include, butare not limited to, monoclonal antibodies, bispecific antibodies,minibodies, domain antibodies, synthetic antibodies (sometimes referredto as antibody mimetics), chimeric antibodies, humanized antibodies,antibody fusions (sometimes referred to as “antibody conjugates”), andfragments of each, respectively. As another example, the analytemolecule may be an antibody and the capture component may be an antigen.

According to one embodiment in which an analyte particle is a biologicalcell (e.g., mammalian, avian, reptilian, other vertebrate, insect,yeast, bacterial, cell, etc.), the capture component may be a ligandhaving specific affinity for a cell surface antigen (e.g., a cellsurface receptor). In one embodiment, the capture component is anadhesion molecule receptor or portion thereof, which has bindingspecificity for a cell adhesion molecule expressed on the surface of atarget cell type. In use, the adhesion molecule receptor binds with anadhesion molecule on the extracellular surface of the target cell,thereby immobilizing or capturing the cell. In one embodiment in whichthe analyte particle is a cell, the capture component is fibronectin,which has specificity for, for example, analyte particles comprisingneural cells.

In some embodiments, as will be appreciated by those of ordinary skillin the art, it is possible to detect analyte molecules using capturecomponents for which binding to analyte molecules is not highlyspecific. For example, such systems/methods may use different capturecomponents such as, for example, a panel of different binding ligands,and detection of any particular analyte molecule is determined via a“signature” of binding to this panel of binding ligands, similar to themanner in which “electronic noses” work. This may find particularutility in the detection of certain small molecule analytes. In someembodiments, the binding affinity between analyte molecules and capturecomponents should be sufficient to remain bound under the conditions ofthe assay, including wash steps to remove molecules or particles thatare non-specifically bound. In some cases, for example in the detectionof certain biomolecules, the binding constant of the analyte molecule toits complementary capture component may be between at least about 10⁴and about 10⁶ M⁻¹, at least about 10⁵ and about 10⁹ M⁻¹, at least about10⁷ and about 10⁹ M⁻¹, greater than about 10⁹ M⁻¹, or the like. Forexample, typical affinities for IgG antibodies for their antigens are inthe range 10⁵-10¹⁰ M⁻¹. The affinity of biotin for streptavidin is 10¹⁵M⁻¹.

In certain embodiments, the capture component is chosen to be able tobind to a corresponding binding partner associated with or attached tothe analyte molecule. For example, the capture component according toone embodiment is a chemical crosslinker as described above able to bindto proteins generally. According to one embodiment, every proteinmolecule in a fluid sample comprises an analyte molecule that attachesto such a chemical crosslinker. In another example, the capturecomponent comprises streptavidin, which binds with high affinity tobiotin, and thus captures any analyte molecules to which biotin has beenattached. Alternatively, the capture component may be biotin, andstreptavidin may be attached to or associated with the analyte moleculessuch that the analyte molecules can be captured by the biotin.

According to one embodiment, the binding surfaces of a capture objectmay be functionalized with capture components in the following manner.First, the surface of a capture object is prepared for attachment of thecapture component(s) by being modified to form or directly bind to thecapture components, or a linker may be added to the binding surface ofthe capture object such that the capture component(s) attaches to thebinding surface of the capture object via the linker. In one embodiment,the binding surfaces of the capture object are derivatized with achemical functionality as described above. Next, the capture componentmay be added, which binds to and is immobilized by the chemicalfunctionality.

Exemplary Target Analytes

As will be appreciated by those in the art, a large number of analytemolecules and particles may be detected and, optionally, quantifiedusing methods and systems of the present invention; basically, anyanalyte molecule that is able to be made to become immobilized withrespect to a capture object (e.g., via a binding surface comprising aplurality of capture components) can be potentially investigated usingthe invention. Certain more specific targets of potential interest thatmay comprise an analyte molecule are mentioned below. The list below isexemplary and non-limiting.

In some embodiments, the analyte molecule may be an enzyme. Non-limitingexamples of enzymes include, an oxidoreductase, transferase, kinase,hydrolase, lyase, isomerase, ligase, and the like. Additional examplesof enzymes include, but are not limited to, polymerases, cathepsins,calpains, amino-transferases such as, for example, AST and ALT,proteases such as, for example, caspases, nucleotide cyclases,transferases, lipases, enzymes associated with heart attacks, and thelike. When a system/method of the present invention is used to detectthe presence of viral or bacterial agents, appropriate target enzymesinclude viral or bacterial polymerases and other such enzymes, includingviral or bacterial proteases, or the like.

In other embodiments, the analyte molecule may comprise an enzymaticcomponent. For example, the analyte particle can be a cell having anenzyme or enzymatic component present on its extracellular surface.Alternatively, the analyte particle is a cell having no enzymaticcomponent on its surface. Such a cell is typically identified using anindirect assaying method described below. Non-limiting example ofenzymatic components are horseradish peroxidase, beta-galactosidase, andalkaline phosphatase.

In yet other embodiments, the analyte molecule may be a biomolecule.Non-limiting examples of biomolecules include hormones, antibodies,cytokines, proteins, nucleic acids, lipids, carbohydrates, lipidscellular membrane antigens and receptors (neural, hormonal, nutrient,and cell surface receptors) or their ligands, or combinations thereof.Non-limiting embodiments of proteins include peptides, polypeptides,protein fragments, protein complexes, fusion proteins, recombinantproteins, phosphoproteins, glycoproteins, lipoproteins, or the like. Aswill be appreciated by those in the art, there are a large number ofpossible proteinaceous analyte molecules that may be detected orevaluated for binding partners using the present invention. In additionto enzymes as discussed above, suitable protein analyte moleculesinclude, but are not limited to, immunoglobulins, hormones, growthfactors, cytokines (many of which serve as ligands for cellularreceptors), cancer markers, etc. Non-limiting examples of biomoleculesinclude PSA and TNF-alpha.

In certain embodiments, the analyte molecule may be ahost-translationally modified protein (e.g., phosphorylation,methylation, glycosylation) and the capture component may be an antibodyspecific to a post-translational modification. Modified proteins may becaptured with capture components comprising a multiplicity of specificantibodies and then the captured proteins may be further bound to abinding ligand comprising a secondary antibody with specificity to apost-translational modification. Alternatively, modified proteins may becaptured with capture components comprising an antibody specific for apost-translational modification and then the captured proteins may befurther bound to binding ligands comprising antibodies specific to eachmodified protein.

In another embodiment, the analyte molecule is a nucleic acid. A nucleicacid may be captured with a complementary nucleic acid fragment (e.g.,an oligonucleotide) and then optionally subsequently labeled with abinding ligand comprising a different complementary oligonucleotide.

Suitable analyte molecules and particles include, but are not limited tosmall molecules (including organic compounds and inorganic compounds),environmental pollutants (including pesticides, insecticides, toxins,etc.), therapeutic molecules (including therapeutic and abused drugs,antibiotics, etc.), biomolecules (including hormones, cytokines,proteins, nucleic acids, lipids, carbohydrates, cellular membraneantigens and receptors (neural, hormonal, nutrient, and cell surfacereceptors) or their ligands, etc), whole cells (including prokaryotic(such as pathogenic bacteria) and eukaryotic cells, including mammaliantumor cells), viruses (including retroviruses, herpesviruses,adenoviruses, lentiviruses, etc.), spores, etc.

The fluid sample containing or suspected of containing an analytemolecule may be derived from any suitable source. In some cases, thesample may comprise a liquid, fluent particulate solid, fluid suspensionof solid particles, supercritical fluid, and/or gas. In some cases, theanalyte molecule may be separated or purified from its source prior todetermination; however, in certain embodiments, an untreated samplecontaining the analyte molecule may be tested directly. The source ofthe analyte molecule may be synthetic (e.g., produced in a laboratory),the environment (e.g., air, soil, etc.), a mammal, an animal, a plant,or any combination thereof. In a particular example, the source of ananalyte molecule is a human bodily substance (e.g., blood, serum,plasma, urine, saliva, tissue, organ, or the like). The volume of thefluid sample analyzed may potentially be any amount within a wide rangeof volumes, depending on a number of factors such as, for example, thenumber of capture objects used/available, the number of locationsus/available, etc. In a few particular exemplary embodiments, the samplevolume may be about 0.01 ul, about 0.1 uL, about 1 uL, about 5 uL, about10 uL, about 100 uL, about 1 mL, about 5 mL, about 10 mL, or the like.In some cases, the volume of the fluid sample is between about 0.01 uLand about 10 mL, between about 0.01 uL and about 1 mL, between about0.01 uL and about 100 uL, or between about 0.1 uL and about 10 uL.

In some cases, the fluid sample may be diluted prior to use in an assay.For example, in embodiments where the source of an analyte molecule is ahuman body fluid (e.g., blood, serum), the fluid may be diluted with anappropriate solvent (e.g., a buffer such as PBS buffer). A fluid samplemay be diluted about 1-fold, about 2-fold, about 3-fold, about 4-fold,about 5-fold, about 6-fold, about 10-fold, or greater, prior to use. Thesample may be added to a solution comprising the plurality of captureobjects, or the plurality of capture objects may be added directly to oras a solution to the sample.

Binding Ligands and Precursor Labeling Agents/Labeling Agent

Binding ligands may be selected from any suitable molecule, particle, orthe like, as discussed more below, able to associate with an analytemolecule and/or to associate with another binding ligand. Certainbinding ligands can comprise an entity that is able to facilitatedetection, either directly (e.g., via a detectable moiety) orindirectly. A component may facilitate indirect detection, for example,by converting a precursor labeling agent molecule into a labeling agentmolecule (e.g., an agent that is detected in an assay). In someembodiments, the binding ligand may comprise an enzymatic component(e.g., horseradish peroxidase, beta-galactosidase, alkaline phosphatase,etc). A first type of binding ligand may or may not be used inconjunction with additional binding ligands (e.g., second type, etc.),as discussed herein.

In some embodiments, the plurality of capture objects, at least some ofwhich comprise at least one analyte molecule, may be exposed to aplurality of binding ligands such that a binding ligand associates withat least some of the plurality of analyte molecules. In embodimentswhere a statistically significant fraction of the capture objects areassociated with a single analyte molecule and a statisticallysignificant fraction of the capture objects are not associated with anyanalyte molecules (e.g., where the number of analyte molecules is lessthan the total number of capture objects), a binding ligand mayassociate with substantially all of the analyte molecules immobilizedwith respect to a capture object. In some cases, greater than about 80%,greater than about 85%, greater than about 90%, greater than about 95%,greater than about 97%, greater than about 98%, greater than about 99%,or more, analyte molecules may become associated with a binding ligand.

In other embodiments where substantially all of the capture objectscomprise at least one analyte molecule (e.g., in embodiments where thenumber of analyte molecules is about equal to or greater than the numberof capture objects provided), the capture objects may be exposed to thebinding ligand such that a statistically significant fraction of thecapture objects associate with at least one binding ligand (or incertain embodiments substantially only a single binding ligand) and astatistically significant fraction of the capture objects do notassociate with any binding ligand. In some cases, the capture objectsmay be exposed to the binding ligands such that at least some of thecapture objects associate with at least one binding ligand and astatistically significant fraction of the capture objects do notassociate with any binding ligand. A screening test to determine anappropriate amount of binding ligand to use for a desired degree ofbinding ligand loading (e.g. to facilitate selection of an appropriatequantity of binding ligand to use for a particular situation) may beperformed with calibration standards containing a known concentration ofanalyte molecule and varying quantities of binding ligand using aPoisson analysis. In certain embodiments, it is determined whether theanalyte is essentially fully labeled or only partially labeled withbinding ligand. The percentage active analyte molecules (i.e. thoseassociated with binding ligand) detected can be converted to thepercentage analyte molecules associated with zero, one, two etc. bindingligands using Poisson distribution adjustment as described elsewhereherein.

In some embodiments, more than one type of binding ligand may be used.In some embodiments, a first type of binding ligand and a second type ofbinding ligand may be provided. In some instances, at least two, atleast three, at least four, at least five, at least eight, at least ten,or more, types of binding ligands may be provided. When a plurality ofcapture objects, some of which are associated with at least one analytemolecule, are exposed to a plurality of types of binding ligand, atleast some of the plurality of immobilized analyte molecules mayassociate with at least one of each type of binding ligand. The bindingligands may be selected such that they interact with each other in avariety of different manners. In a first example, the first type ofbinding ligand may be able to associate with an analyte molecule and thesecond type of binding ligand may be able to associate with the firsttype of binding ligand. In such embodiments, the first type of bindingligand may comprise a first component which aids in association of theanalyte molecule and a second component which aids in association of thesecond type of binding ligand with the first type of binding ligand. Ina particular embodiment, the second component is biotin and the secondtype of binding ligand comprises an enzyme or an enzymatic componentwhich associates with the biotin.

As another example, both the first type of binding ligand and the secondtype of binding ligand may associate directly with an analyte molecule.Without being bound by theory or any particular mechanism, theassociation of both the first type and the second type of binding ligandmay provide additional specificity and reliability in performing anassay, by identifying only locations which are determined to containboth the first type of binding ligand and/or the second type of bindingligand (e.g., either through direct or indirect detection) as containingan analyte molecule. Such assay methods may reduce the number of falsepositives caused by non-specific binding as locations that are found toonly have a single type of binding ligand (e.g., only the first type oflabeling agent or the second type of labeling agent) would be not beconsidered or counted as a location comprising an analyte molecule. Thefirst type of binding ligand may comprise a first type of enzymaticcomponent and the second type of binding ligand may comprise a secondtype of enzymatic component which differs from the first type ofenzymatic component. A capture object comprising an analyte molecule,the first type of binding ligand, and the second type of binding ligandmay be exposed to a first type of precursor labeling agent which isconverted to a first type of labeling agent (e.g., comprising a firstmeasurable property) upon exposure to the first type of enzymaticcomponent and a second type of precursor labeling agent which isconverted to a second type of labeling agent (e.g., comprising ameasurable property which is distinguishable from the first measurableproperty) upon exposure to the second type of enzymatic component.Therefore, only locations which are determined to contain the first typeof labeling agent and the second type of labeling agent are determinedto contain an analyte molecule. As another example, the first type ofbinding ligand and the second type of binding ligand may eachincorporate a component (e.g., such as a DNA label) and a third type ofbinding ligand may comprise two components complimentary to thecomponents of the first type and second type of binding ligands (e.g.,two types of complimentary DNA labels), wherein the third type ofbinding ligand also comprises an molecule or moiety for direct orindirect detection (e.g., the presence of the third type of bindingligand in a reaction vessel is required to determine the presence orabsence of an analyte molecule in a location). When both the first typeof binding ligands and the second types of binding ligands are presentin substantially close proximity to each other (e.g., via associationwith an analyte molecule) association of the third type of bindingligand may occur, thus allowing detection of the analyte molecule. Moreinformation regarding the use of more than one type of binding ligand ina manner which may reduce certain negative affects associated withnon-specific binding, are described in commonly owned U.S. patentapplication Ser. No. ______ (not yet determined), entitled“Ultra-Sensitive Detection of Molecules using Dual Detection Methods” byDuffy et al., filed Mar. 24, 2010 (Attorney Docket No. Q0052.70012US01),incorporated by reference.

Detection

The plurality of capture objects, some of which comprise at least oneanalyte molecule and/or at least one binding ligand can be detectedand/or quantified, and the detection and/or quantification can berelated to the presence and, optionally, the quantity and/orconcentration of analyte molecules/particles in the sample being tested.In some embodiments, the plurality of capture objects may be detectedand/or quantified by spatially segregating the plurality of captureobjects into a plurality of locations. In some embodiments, theplurality of locations comprises a plurality of reaction vessels (e.g.,in an array). In some cases, a detector may be configured to detect thecapture objects in or at a plurality of locations (e.g., an array ofreaction vessels). In some embodiments, the capture objects may be ableto produce or be made to produce a detectable signal, for example,fluorescence emission, which may aid in the detection of the captureobjects. In some cases, the capture objects may be detected usingscattering techniques, as described herein.

In some embodiments, the number of capture objects spatially segregatedmay be substantially equal to the number of capture objects exposed to afluid sample containing or suspected of containing analyte molecules. Insome embodiments, however, the number of capture objects spatiallysegregated into a plurality of locations may be substantially less thanthe number of capture objects exposed to a fluid sample containing orsuspected of containing analyte molecules. In some cases, about 1%,about 2%, about 3%, about 5%, about 10%, about 15%, about 20%, about30%, about 40%, about 50%, about 60%, about 70%, about 80%, or more, ofthe capture objects exposed to a fluid sample are spatially segregatedinto a plurality of locations. In some instances, between about 1% andabout 99%, between about 10% and about 90%, between about 20% and about80%, between about 30% and about 70%, between about 50% and about 90%,between about 1% and about 50%, between about 5% and about 40%, orbetween about 10% and about 30% of the capture objects exposed to afluid sample are spatially segregated into a plurality of locations.

The analyte molecules (or binding ligands) which are spatiallysegregated may be detected and/or quantified directly or indirectly. Inthe case of direct detection, the analyte molecules may comprise amolecule or moiety that may be directly interrogated and/or detected,for example, a fluorescent entity (e.g., a fluorescent moiety,fluorescent bead, fluorescent antibody, etc.), a metal nanoparticle ornanocluster (e.g., a gold nanocluster or nanoparticle, silvernanocluster or nanoparticle), a quantum dot (e.g., CdSe quantum dot,CdTe quantum dot, etc.), and radioactive isotopes. In embodiments wherethe assay comprises the use of one or more types of binding ligands, thebinding ligands may comprise a molecule(s) or moiety(ies) that may bedirectly interrogated and/or detected. A location that comprises such ananalyte molecule or binding ligand which comprises a moiety that may bedirectly interrogated and/or detected can be made to emit a signal uponinterrogation of the location.

In some embodiments, non-enzymatic detection methods may be employed.Non-enzymatic detection methods will be known to those of ordinary skillin the art. Non-limiting examples include Raman scattering,electromagnetic radiation resonance methods (e.g., whispering gallerymodes), spectroscopy (e.g., infrared, atomic spectroscopies),absorbance, piezoelectric transduction (e.g., quartz crystalmicrobalance (QCM)), circular dichroism, electron microscopies (e.g.,scanning electron microscopy (SEM), x-ray photoelectron microscopy(XPS)), scanning probe microscopies (e.g., atomic force microscopy(AFM), scanning tunneling microscopy (STM)), light scattering; surfaceplasmon resonance (SPR), evanescent wave detection, opticalinterferometry and other methods based on measuring changes inrefractive index, electrical transduction methods, such as conductionand capacitance; magnetic transduction effects (e.g., magnetoresistiveeffect), calorimetry (e.g., differential scanning calorimetry (DSC)),diffraction; nuclear magnetic resonance (NMR), electron paramagneticresonance (EPR), mass spectroscopy (e.g., matrix assisted laserdesorption and ionization (MALDI)), fluorescence technologies (e.g.,fluorescence resonance energy transfer (FRET), time-resolvedfluorescence (TRF), fluorescence polarization (FP)), and luminescentoxygen channeling (LOCI).

In some embodiments, the plurality of analyte molecules (or bindingligands) are indirectly detected. The indirect approach can include, forexample, exposing an analyte molecule, or a binding ligand associatedwith an analyte molecule, to a precursor labeling agent, wherein theprecursor labeling agent is converted into a labeling agent uponexposure to the analyte molecule or the binding ligand associated withan analyte molecule. The labeling agent may comprise a molecule ormoiety that can be interrogated and/or detected. The presence or absenceof an analyte molecule or binding ligand at a location may then bedetermined by determining the presence or absence of a labeling agentat/in the location. For example, the analyte molecule may comprise anenzymatic component and the precursor labeling agent molecule may be achromogenic, fluorogenic, or chemiluminescent enzymatic precursorlabeling agent molecule which is converted to a chromogenic,fluorogenic, or chemiluminescent product (each an example of a labelingagent) upon exposure to the converting agent. In this instance, theprecursor labeling agent may be an enzymatic label, for example, achromogenic, fluorogenic, or chemiluminescent enzymatic precursorlabeling agent, that upon contact with the enzymatic component, isconverted into a labeling agent, which is detectable. In some cases, thechromogenic, fluorogenic, or chemiluminescent enzymatic precursorlabeling agent is provided in an amount sufficient to contact everylocation. In some embodiments, an electrochemiluminescent precursorlabeling agent is converted to an electrochemiluminescent labelingagent. In some cases, the enzymatic component may comprisebeta-galactosidase, horseradish peroxidase, or alkaline phosphatase.

As will be understood by those of ordinary skill in the art, a varietyof appropriate chromogenic, fluorogenic, or chemiluminescent enzymaticprecursor labeling agents may be selected for conversion by manydifferent enzymes. Thus, any known chromogenic, fluorogenic, orchemiluminescent enzyme precursor labeling agent capable of producing alabeling agent in a reaction with a particular enzyme can potentially beused in the present invention as the precursor labeling agent inembodiments where the analyte molecule or a binding ligand associatedwith an analyte molecule comprises an enzymatic component. For example,many chromogenic, fluorogenic, or chemiluminescent precursor labelingagent suitable for use an enzymatic precursor labeling agent moleculeare disclosed in The Handbook—A Guide to Fluorescent Probes and LabelingTechnologies, Tenth Ed., Chapter 10.

In another embodiment, the analyte molecule may be a protein and thebinding ligand may comprise a component which is capable of binding bothto the analyte molecule and an enzymatic component. Exposure of theprecursor labeling agent molecule to the enzymatic component bound tothe binding ligand may convert the precursor labeling agent molecule toa chromogenic, fluorogenic, of chemiluminescent labeling agent moleculethat may be detected.

Two non-limiting examples of indirect detection of an analyte moleculeare illustrated in FIGS. 9A and 9B. In FIG. 9A, a location 150 (in thisembodiment, represented by a reaction vessel) is provided whichcomprises capture object 152 (in this embodiment, represented by abead). Analyte molecule 154 is immobilized with respect to captureobject 152 via capture component 156. The reaction vessel is exposed toprecursor labeling agent 158, which upon exposure to analyte molecule154, is converted to labeling agent molecule 160, as indicated by arrow159. As another example, in FIG. 9B, location 170 (in this embodiment,represented by a reaction vessel) is provided which comprises captureobject 172 (in this embodiment, represented by a bead). Analyte molecule174 is immobilized with respect to capture object 172 via capturecomponent 176, and binding ligand 177 is associated with analytemolecule 174. The reaction vessel is exposed to precursor labeling agent158, which upon exposure to binding ligand 177, is converted to alabeling agent molecule 180, as indicated by arrow 179.

In some embodiments, a plurality of locations may be addressed and/or aplurality of capture objects and/or species/molecules/particles ofinterest may be detected substantially simultaneously. “Substantiallysimultaneously” when used in this context, refers toaddressing/detection of the locations/captureobjects/species/molecules/particles of interest at approximately thesame time such that the time periods during which at least twolocations/capture objects/species/molecules/particles of interest areaddressed/detected overlap, as opposed to being sequentiallyaddressed/detected, where they would not. Simultaneousaddressing/detection can be accomplished by using various techniques,including optical techniques (e.g., CCD detector). Spatially segregatingcapture objects/species/molecules/particles into a plurality ofdiscrete, resolvable locations, according to some embodimentsfacilitates substantially simultaneous detection by allowing multiplelocations to be addressed substantially simultaneously. For example, forembodiments where individual species/molecules/particles are associatedwith capture objects that are spatially segregated with respect to theother capture objects into a plurality of discrete, separatelyresolvable locations during detection, substantially simultaneouslyaddressing the plurality of discrete, separately resolvable locationspermits individual capture objects, and thus individualspecies/molecules/particles (e.g., analyte molecules) to be resolved.For example, in certain embodiments, individual molecules/particles of aplurality of molecules/particles are partitioned across a plurality ofreaction vessels such that each reaction vessel contains zero or onlyone species/molecule/particle. In some cases, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, atleast about 99.5% of all species/molecules/particles are spatiallyseparated with respect to other species/molecules/particles duringdetection. A plurality of species/molecules/particles may be detectedsubstantially simultaneously within a time period of less than about 1second, less than about 500 milliseconds, less than about 100milliseconds, less than about 50 milliseconds, less than about 10milliseconds, less than about 1 millisecond, less than about 500microseconds, less than about 100 microseconds, less than about 50microseconds, less than about 10 microseconds, less than about 1microsecond, less than about 0.5 microseconds, less than about 0.1microseconds, or less than about 0.01 microseconds, less than about0.001 microseconds, or less. In some embodiments, the plurality ofspecies/molecules/particles may be detected substantially simultaneouslywithin a time period of between about 100 microseconds and about 0.001microseconds, between about 10 microseconds and about 0.01 microseconds,or less.

During the step of the method where the locations into which the captureobjects/analyte molecules have been segregated are addressed, any of avariety of parameters may be determined. In some embodiments, the numberof locations which comprise a capture object and an analyte molecule (orbinding ligand) is determined. The number of locations which comprise acapture object but do not comprise an analyte molecule (or bindingligand) may also be determined. In some cases, the number of locationswhich are addressed which do not contain a capture object may also bedetermined. In still yet other cases, the total number of locationsaddressed may also be determined. A single interrogation or multipleinterrogations of any subset or all of the locations ultimatelyaddressed may be made at any given time to facilitate one or all of theabove described determinations. For example, a first determination maybe completed under a first range of wavelengths (e.g., white light) todetermine the number of locations comprising a capture object, whereinthe locations are not distinguished as to whether an analyte molecule(or binding ligand) is associated with the capture object, and a seconddetermination of the same or some subset of the locations may becompleted under a second range of wavelengths (e.g., fluorescence) todetermine the number of locations which comprise a capture objectassociated with an analyte molecule (or binding ligand). Exemplarydetection methods are described below.

Detection Methods

In some embodiments, in the systems/methods in which the species to bedetected are partitioned across a plurality of locations, the locationscan be interrogated using a variety of techniques, including techniquesknown to those of ordinary skill in the art.

In a specific embodiment of the present invention, the locations areoptically interrogated. The locations exhibiting changes in theiroptical signature may be identified by a conventional optical train andoptical detection system. Depending on the detected species (e.g.,labeling agent molecules, particles, etc.) and the operativewavelengths, optical filters designed for a particular wavelength may beemployed for optical interrogation of the locations.

In embodiments where optical interrogation is used, the system maycomprise more than one light source and/or a plurality of filters toadjust the wavelength and/or intensity of the light source. For example,in some cases, a first interrogation of the locations may be conductedusing light of a first range of wavelengths (e.g., white light inembodiments where the capture objects are not fluorescent, or awavelength range where the capture objects fluoresce), whereas a secondinterrogation is conducted using light of a second, differing range ofwavelengths, such that the plurality of detectable molecules fluoresce.An exemplary system configuration is provided below (see FIG. 10).

In some embodiments, the optical signal from a plurality of locations iscaptured using a CCD camera Other non-limiting examples of cameraimaging types that can be used to capture images include chargeinjection devices (CIDs), complimentary metal oxide semiconductors(CMOSs) devices, scientific CMOS (sCMOS) devices, and time delayintegration (TDI) devices, as will be known to those of ordinary skillin the art. The camera may be obtained from a commercial source. CIDsare solid state, two dimensional multi pixel imaging devices similar toCCDs, but differ in how the image is captured and read. For examples ofCIDs, see U.S. Pat. No. 3,521,244 and U.S. Pat. No. 4,016,550. CMOSdevices are also two dimensional, solid state imaging devices but differfrom standard CCD arrays in how the charge is collected and read out.The pixels are built into a semiconductor technology platform thatmanufactures CMOS transistors thus allowing a significant gain in signalfrom substantial readout electronics and significant correctionelectronics built onto the device. For example, see U.S. Pat. No.5,883,830. sCMOS devices comprise CMOS imaging technology with certaintechnological improvements that allows excellent sensitivity and dynamicrange. TDI devices employs a CCD device that allows columns of pixels tobe shifted into and adjacent column and allowed to continue gatheringlight. This type of device is typically used in such a manner that theshifting of the column of pixels is synchronous with the motion of theimage being gathered such that a moving image can be integrated for asignificant amount of time and is not blurred by the relative motion ofthe image on the camera. In some embodiments, a scanning mirror systemcoupled with a photodiode or photomultiplier tube (PMT) could be used tofor imaging.

In one embodiment, the plurality of locations is formed directly as aplurality of reaction vessels in an end of a fiber optic bundle.According to one embodiment, the array of reaction vessels for thepresent invention can be used in conjunction with an optical detectionsystem such as the system described in U.S. Publication No.2003/0027126. For example, according to one embodiment, the array ofreaction vessels of the present invention is formed in one end of afiber optic assembly comprising a fiber optic bundle constructed of cladfibers so that light does not mix between fibers.

FIGS. 10A and 10B show non-limiting examples of a system of the presentinvention according to some embodiments. The system comprises a lightsource 452, excitation filter 454, dichromatic mirror 458, emissionfilter 460, objective 470, and array 472. Light 453 given off from lightsource 452 is passed through excitation filter 454. The light reflectsoff dichromatic mirror 458, passes through objective 470 and shines onarray 472. In some cases, stray light 464 may be reduced by a straylight reducing function 468, such as an iris or aperture. Light 471emitted from the array passes through objective 470 and emission filter460. Light 462 is observed. The system may comprise additionalcomponents (e.g., additional filters, mirrors, magnification devices,etc.) as needed for particular applications, as would be understood bythose of ordinary skill in the art.

The system shown in FIG. 10A may additionally comprise components whichaid in the determination of the number of reaction vessels which containa capture object (e.g., using white light). Alternatively, theadditional components may be used to determine the total number oflocations and/or provide spatially information regarding the position ofthe locations (e.g., containing or not containing a capture object),which may help corroborate signals observed under different lightregimes (e.g., fluorescence, white light) corresponding with theposition of a location (e.g., a mask may be created).

In FIGS. 10A and 10B, excitation light is emitted from source 452 andcollimated into a beam 453. The excitation filter 454 may be configuredto transmit only the wavelength band that excites the fluorophore (e.g.,575 nm+/−10 nm for resorufin). The excitation light is reflecteddownward by the dichroic filter 458 and excites the substrate 472containing the sample through the objective lens 470. The image light iscollected by the objective lens 470, collimated into a beam 471 andtransmitted through the dichroic filter 458. Only the image lightcorresponding to the fluorescence wavelength band (e.g., 670 nm+/−30 nmfor resorufin) is transmitted through the emission filter 460. Theremaining collimated beam 462 contains only the emitted fluorescencewavelengths which will subsequently be imaged through the camera system.

The same system may be used to determine the positioning of thelocations containing sample (e.g., reaction vessels). The arraycomprising the reaction vessels containing capture objects may beilluminated with a “bright field” white light illumination. The arraymay be illuminated (e.g., using light source 475 shown in FIG. 10A) bydirecting a pseudo-collimated white light (e.g., white light LED) ontothe array surface from an angle (e.g., θ₁ in FIG. 10A may be about 20degrees, about 25 degrees, about 30 degrees, about 35 degrees, about 40degrees, or greater) just outside the numerical aperture of thecollection objective. Light that hits the surface of the array 472(e.g., light 476) is reflected (and scattered) off the surface,collimated 471, and collected by the objective lens (470). Thecollimated beam is subsequently imaged through the camera system.

The same system may also be used to determine which locations contain acapture object (e.g., bead). Any particular bead may or may not beassociated with an analyte molecule and/or binding ligand. The array maybe illuminated (e.g., using light source 473 as shown in FIG. 10A) witha “dark field” white light illumination. The array may be illuminated byaiming a pseudo-collimated white light (e.g., white light LED 473) ontothe array surface from an angle (e.g., θ₂ in FIG. 10A is about 65degrees, about 70 degrees, about 75 degrees, about 80 degrees, about 85degrees) substantially outside the numerical aperture of the collectionobjective. Light that hits the surface of the array 472 (e.g., light474) is reflected (and scattered) off the surface, collimated 471, andcollected by the objective lens 470. The collimated beam is subsequentlyimaged by the camera system.

In some embodiments, an optical detection system may be employed, forexample, as described in U.S. Publication No. 2003/0027126. In anexemplary system, light returning from an array of reaction vesselsformed at the distal end of a fiber optic bundle is altered via use of amagnification changer to enable adjustment of the image size of thefiber's proximal or distal end. The magnified image is then shutteredand filtered by a shutter wheel. The image is then captured by chargecoupled device (CCD) camera. A computer may be provided that includesand executes imaging processing software to process the information fromthe CCD camera and also optionally may be configured to control shutterand filter wheels. As depicted in U.S. Publication No. 20030027126, theproximal end of the bundle is received by a z-translation stage and x-ymicropositioner.

For example, FIG. 11 shows a schematic block diagram of a systememploying a fiber optic assembly 400 with an optical detection system.The fiber optic assembly 400 that comprises a fiber optic bundle orarray 402 that is constructed from clad fibers so that light does notmix between fibers. An array of reaction vessels 401 is formedat/attached to the bundle's distal end 412, with the proximal end 414being operatively connected with a z-translation stage 416 and x-ymicropositioner 418. These two components act in concert to properlyposition the proximal end 414 of the bundle 402 for a microscopeobjective lens 420. Light collected by the objective lens 420 is passedto a reflected light fluorescence attachment with three pointer cubeslider 422. The attachment 422 allows directs light from a 75 watt Xelamp 424 through the objective lens 420 to be coupled into the fiberbundle 402. The light from source 424 is condensed by condensing lens426, then filtered and/or shuttered by filter and shutter wheel 428, andsubsequently passes through a ND filter slide 430. Light returning fromthe distal end 412 of the bundle 402 passes through the attachment 422to a magnification changer 432 which enables adjustment of the imagesize of the fiber's proximal or distal end. Light passing through themagnification changer 432 is then shuttered and filtered by a secondwheel 434. The light is collected by a charge coupled device (CCD)camera 436. A computer 438 executes imaging processing software toprocess the information from the CCD camera 436 and also optionallycontrols other components of the system, including but not limited tothe first and second shutter and filter wheels 428, 434.

An array of reaction vessels used to practice some embodiments of thepresent invention may be integral with or attached to the distal end ofthe fiber optic bundle using a variety of compatible processes. In somecases, microwells are formed at the center of each individual fiber ofthe fiber optic bundle and the microwells may or may not be sealed. Eachoptical fiber of the fiber optic bundle may convey light from the singlemicrowell formed at the center of the fiber's distal end. This featureenables the interrogation of the optical signature of individualreaction vessels to identify reactions/contents in each microwell.Consequently, by collecting the image of the end of the bundle with theCCD array, the optical signatures of the reaction vessels may beindividually interrogated and/or imaged substantially simultaneously.

Quantification

According to some embodiments of the present invention, the methods,systems, and/or devices are used to determine the presence and/or ameasure of the concentration of analyte molecules (or particles) in afluid sample based at least in part on detecting and/or quantifying atleast some of a plurality of capture objects used to capture the analytemolecules (and optionally at least one binding ligand). In certainembodiments where concentration is determined, a correlation and/orcalibration relating the number (or fraction/percentage) of locationscontaining a capture object comprising at least one analyte molecule(and/or at least one binding ligand) to the quantity/concentration ofanalyte molecules in the fluid sample is employed. In some cases, theconcentration of the analyte molecules in a fluid sample may be linearlyproportional to the number/fraction of locations containing a captureobject comprising at least one analyte molecule (and/or at least onebinding ligand). In other cases, the measure of concentration of theanalyte molecules in a fluid sample may be related to thenumber/fraction of locations containing a capture object associated withat least one analyte molecule (and/or at least one binding ligand) by anon-linear relationship. In some embodiments, a measure of theconcentration of analyte molecules in a fluid sample may be determinedat least in part using a calibration curve developed using samplescontaining known concentrations of target analyte molecules. Methods todetermine a measure of the concentration of analyte molecules in a fluidsample are discussed more below.

Certain embodiments of present invention are distinguished by theability to detect and/or quantify low numbers/concentrations of captureobjects comprising at least one analyte molecule (and/or at least onebinding ligand) and may be well suited to determine a measure of theconcentration of analyte molecules in a fluid sample containing very lowconcentrations of the analyte molecule. This capability may befacilitated, in certain embodiments, at least in part by spatiallyisolating individual capture objects, including at least some comprisingat least one analyte molecule (and/or at least one binding ligand), forexample, by partitioning a plurality of such capture objects across anarray of locations (e.g., reaction vessels), and then detecting theirpresence in the reaction vessels. The presence of a capture objectcomprising at least one analyte molecule (and/or at least one bindingligand) in a reaction vessel, in some embodiments, can be determined andthe number of such reaction vessels counted in a binary fashion. Thatis, in embodiments where a location (e.g., a reaction vessel) is foundto contain at least one capture object associated with at least oneanalyte molecule (and/or binding ligand), the location is counted asone. In embodiments where a location (e.g., a reaction vessel) is foundto contain a capture object, the location is counted as zero. Forexample, wells that are counted as “ones” may be determined by detectingthe presence of a detectable molecule or particle in a reaction vesselthat, as described above, indicates the presence of a capture objectcomprising at least one analyte molecule (and/or at least one bindingligand) in the well.

In embodiments where a fluid sample containing or suspected ofcontaining is contacted with a plurality of capture objects such thatany analyte molecules present in the sample are immobilized with respectto the plurality of capture objects such that a statisticallysignificant fraction (e.g., as described above) of the capture objectsassociate with a single analyte molecule and a statistically significantfraction of the capture objects do not associate with any analytemolecules (e.g., as shown in FIG. 1, step (B)), a determination of ameasure of the concentration of analyte molecules in the fluid samplemay be carried out as follows. First, at least a portion of the captureobjects (at least some of which have a single analyte moleculeimmobilized) are spatially segregated into a plurality of locations(e.g., as shown in FIG. 1, step (C)). The number of locations thatcontain an analyte molecule immobilized with respect to a capture objectis determined, either directly (e.g., by detection of the analytemolecule itself (e.g., see FIG. 1, step (D)) or indirectly (e.g., bydetection of a binding ligand associated with the analyte molecule, bydetection of a labeling agent (e.g., formed via conversion of aprecursor labeling agent upon exposure to an analyte molecule see FIG.4A), etc.). In some embodiments, a measure of the concentration ofanalyte molecules in a fluid sample is determined at least in part onthe determination of the number of the plurality of locations thatcontain an analyte molecule (e.g., reaction vessels 12 in FIG. 1, step(D)). In certain such embodiments, a measure of the concentration ofanalyte molecules in the fluid sample is determined at least in part bycomparison of this measured parameter to a calibration standard and/orby using a Poisson and/or Gaussian distribution analysis of the numberof locations that would be expected to contain an analyte molecule.

In some embodiments the number of locations which comprise a captureobject not associated with an analyte molecule may also be determined(e.g., reaction vessel 13 in FIG. 1, step (D)). In such cases, a measureof the concentration of analyte molecules in a fluid sample may bedetermined based at least in part on the ratio of locations comprisingan analyte molecule immobilized with respect to a capture object, to thenumber of locations comprising a capture object not associated with ananalyte molecule. In some cases, the number of locations which do notcomprise a capture object may also be determined (e.g., reaction vessel14 in FIG. 1, step (D)). In such cases, a measure of the concentrationof analyte molecules in a fluid sample may be determined based at leastin part on the ratio of locations comprising an analyte moleculeimmobilized with respect to a capture object to the number of locationsnot comprising a capture object and/or the number of locations notcomprising an analyte molecule—whether or not such location contains acapture object (in either case above or elsewhere, the denominator forthe ratio/fraction may or may not include the positive (“on” or “one”)locations added to the nil (“off” or “zero”) locations depending uponpreference). In yet other cases, the total number of locationsaddressed/analyzed may be determined (e.g., reaction vessels 12, 13, and14 in FIG. 1, step (D)) and a measure of the concentration of analytemolecules in a fluid sample may be based on the ratio of the locationscomprising an analyte molecule immobilized with respect to a captureobject to the total number of locations addressed/analyzed.

It should be understood, that in some assay methods, a measure of theconcentration of analyte molecules in a fluid sample may be carried outusing more than one type of analysis (e.g., a first analysis based onthe number of locations comprising an analyte molecule immobilized withrespect to a capture object, and a second analysis based on the ratio ofthe ratio/fraction of locations comprising an analyte moleculeimmobilized with respect to a capture component, to the total number oflocations comprising a capture object, etc.). In such embodiments, thesecond analysis may be used as a quality control measure (e.g., toconfirm that the first analysis provided a reasonable result) and/or thetwo analysis results may be averaged.

In some embodiments, the determination of a measure of the concentrationof analyte molecules in a fluid sample being tested may be carried outusing a similar analysis as described above, but by determining thenumber of reaction vessels which comprise a binding ligand as opposed tothe number of reaction vessels which comprise an analyte moleculeimmobilized with respect to a capture object. As described herein, insome cases, following immobilization of a plurality of analyte moleculesto a plurality of capture objects, the plurality of capture objects maybe exposed to at least one type of binding ligand such that at leastsome of the immobilized analyte molecules associate with at least onebinding ligand (e.g., see FIG. 2, step (B). This assay method may beespecially useful in embodiments where more than one analyte molecule isexpected to become associated with each capture object, but binaryquantification may still be desired. In some cases, the binding ligandmay be provided at a concentration such that at least some of thecapture objects containing at least one analyte molecule do notassociate with any binding ligands (e.g., see FIG. 2, step (C). In suchembodiments, the number of locations containing a capture objectassociated with a binding ligand (e.g., via an analyte molecule) canreplace the number of locations containing a capture object associatedwith an analyte molecule in the analysis and methods described above.

A measure of the concentration of analyte molecules or particles in afluid sample may be determined using a variety of calibrationtechniques, and the particular technique resulting in the most accuracyand reliability can depend on the relative number/concentration ofanalyte molecules in the sample to the number/concentration of captureobjects exposed to the sample (and or, for embodiments using bindingligands, the relative number/concentration of binding ligands to thenumber/concentration of capture objects exposed to each otherduring/after capture of the analyte molecules by the capture objects).Non-limiting examples of concentration determination methods that may beuseful in particular analyte concentration regimes include the abovedescribed binary read-out methods, and/or methods in which the relativepositive signal intensity measured for the locations (“intensityread-out methods”) is employed. Either or both of the above methods—oralternative methods—may further employ a comparison of the measuredparameter with a calibration curve.

It is currently believed that the most accurate method of determinationmay depend at least in part on the concentration of analyte moleculescontained in the fluid sample. For example, in embodiments in which theconcentration of the analyte molecules in the sample being testedresults in a statistically significant fraction of locations to whichthat capture objects are partitioned comprising a single analytemolecule or binding ligand and a statistically significant fraction oflocations not comprising any analyte molecules or binding ligands (e.g.,at or approaching a regime where essentially no locations comprise morethan one analyte molecule or binding ligand), a binary read-out methodmay be particularly useful, and in some cases, may be used inconjunction with a calibration curve. In other embodiments, where alarger number of locations comprise more than one analyte moleculeand/or more than one binding ligand, a determination based at least inpart on an intensity read-out may provide a more accurate measure of theconcentration of analyte molecules in a fluid sample. Such adetermination may also be used in conjunction with a calibration curve.

In certain embodiments, the fraction of locations (e.g., thestatistically significant fraction) which comprises at least one captureobject associated with an analyte molecule and/or binding ligand is lessthan about 50%, less than about 40%, less than about 25%, less thanabout 10%, less than about 5%, less than about 1%, less than about 0.5%,or less than about 0.1% of the total number of locations containing acapture object. In such embodiments, a measure of the concentration ofanalyte molecules in the fluid sample may be determined using a binaryread-out method. In some cases, the percentage of locations which do notcontain a capture object associated with an analyte molecule and/orbinding ligand is at least about 20%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 75%, atleast about 80%, at least about 90%, or at least about 95%, at leastabout 99%, at least about 99.5%, at least about 99.9%, or greater, ofthe total number of locations.

While the discussion below focuses primarily on the use of a binaryread-out system (e.g., based on counting the number of “on” and “off”locations) for ultra low level detection capability, this is by no meanslimiting, and the inventive methods and assays may also in certainembodiments employ instead of or in addition to a binary quantificationprotocol, one based in measurement of intensity (i.e. an intensityread-out method) (e.g., to extend dynamic range). As noted, in somecases, the detection systems and quantification methods may beconfigured so that the system can use either or both of a binaryread-out determination and an intensity read-out determination,depending on the assay format and/or the concentration of analytemolecules in the fluid sample. For example, the method and/or system maybe able to determine a base parameter from a first measurement anddecide to use either a binary read-out determination or an intensityread-out determination depending on the result of the firstdetermination, as described in more detail below and as is described incommonly owned U.S. patent application Ser. No. ______ (not yetdetermined), entitled “Methods and systems for extending dynamic rangein assays for the detection of molecules or particles” by Rissin et al.,filed Mar. 24, 2010 (Attorney Docket No. Q0052.70013US01), incorporatedby reference.

According to one embodiment, the quantification method of the presentinvention can be performed as follows. A fluid sample containing orsuspected of containing an analyte molecule of interest is contactedwith a plurality of capture objects and, optionally, one or more bindingligands and the capture objects are partitioned across an array oflocations, such as reaction vessels/wells (as described previously). Insome embodiments, where a binary read-out method is desired to be usedfor determination, in the step of contacting the fluid sample with thecapture objects, the relative amounts/concentrations of fluid sample andcapture object containing solution are selected (e.g., based on a knownor estimated/suspected approximate concentration range of analytemolecules in the sample) so that the ratio of analyte molecules in thefluid sample to total number of capture objects provided to the solutionwill be less than about 1:5, less than about 1:10, less than about 1:12,less than about 1:15, less than about 1:20, less than about 1:50, lessthan about 1:100, or less. With such ratios, at least some of thecapture objects statistically will be expected associate with a singleanalyte molecule and the majority of the remainder of the captureobjects will not associate with any analyte molecules. The number ofcapture objects associating with multiple analyte molecules under suchconditions may be low enough to be neglected, such that capture objectdetermined to comprise an analyte molecule can be assumed to comprise asingle analyte molecule. Under such conditions, an analysis systemconfigured to perform a binary read out quantification may be used todetermine the number of locations which comprise a capture objectassociated with an analyte molecule by any detection method as describedherein. The number of locations which comprise a capture objectassociated with an analyte molecule is then counted (e.g., FIG. 1, step(D), the total number of reaction vessels comprising an analytemolecules is two, e.g., reaction vessels 12) and, in some cases, thefraction of the total number of locations containing a capture objectwhich contain a capture object associated with an analyte molecule iscalculated (e.g., in FIG. 1, total number of reaction vessels comprisinga capture object is three, reactions vessels 12 and 13; thus, fractionof the total number of locations comprising a capture object associatedwith an analyte molecule is 2:3). Utilization of a zero (no analytemolecule detected) or one (an analyte molecule detected) response, inconjunction with using an array with a large number of locations canpermit a determination of bulk concentrations of analyte molecules inthe sample by counting the actual number of molecules contained in thevolume of sample partitioned across and contained in the locations. Insome cases, the analyte molecule may be detected indirectly (e.g., theread-out is accomplished by counting the number of locations containingat least one labeling agent molecule, wherein the labeling agent hasbeen converted from a precursor labeling agent upon exposure to ananalyte molecule). In instances where a large number of locations (e.g.,at least about 10,000 locations) are substantially simultaneouslyinterrogated, the ratio of locations comprising an analyte moleculeassociated with a capture object to total number of locations determined(e.g., in some cases, the locations which contain a capture objectassociate with or not associated with any analyte molecules) may be atleast about 1:100, at least about 1:1000, at least about 1:10,000 orless. Utilizing an array with a large number of locations (e.g., atleast about 10,000, at least about 50,000, at least about 100,000, atleast about 500,000, etc.) may provide a statistically significantsignal even at this low ratio.

In some assays, a Poisson distribution adjustment may be applied tonumbers and/or ratios determined by a binary read-out method tofacilitate and/or improve accuracy of determining a concentration ofanalyte molecules in a fluid sample. For example, in embodiments wherethe ratio of analyte molecules in the fluid sample to the total numberof capture objects contacted with the fluid sample is greater than about1:10, greater than about 1:5, greater than about 1:4, greater than about1:3, or greater than about 1:2, or between about 1:10 and about 1:2,between about 1:5 and about 1:2, the number of analyte moleculesimmobilized per capture may be zero or one, with a greater proportioncontaining more than one than for the regime described in the paragraphabove. In some such cases, performance and accuracy of the concentrationdeterminations may be improved over use of an assumption that allpositive locations contain only a single analyte molecule (as describedin the paragraph above) by employing a Poisson distribution adjustmentto predict the number of locations expected to contain 0, 1, 2, 3, 4,etc., analyte molecules per capture object.

A Poisson distribution describes the likelihood of a number of eventsoccurring if the average number of events is known. If the expectednumber of occurrences is μ, then the probability (P_(μ)(ν)) that thereare exactly ν occurrences (ν being a non-negative integer, ν=0, 1, 2, .. . ) may be determined by Equation 5:

$\begin{matrix}{{P_{\mu}(v)} = {e^{- \mu}\left( \frac{\mu^{v}}{v!} \right)}} & \left( {{Eq}.\mspace{14mu} 5} \right)\end{matrix}$

In some embodiment of the present invention, μ is equal to the fractionof the number of locations determined to contain an analyte moleculeassociated with an analyte to the total number of capture objectsdetected (e.g., either associated with or not associated with anyanalyte molecules), and v is the number of capture objects associatedwith a certain number of analyte molecules (e.g., the number of captureobjects associated with either 0, 1, 2, 3, etc. analyte molecule). Bydetermining μ from interrogating the array of locations during an assay,the concentration of analyte molecules in the sample can be determinedusing a Poisson distribution adjustment. For example, in an assay usingthe binary mode of measurements where capture objects associated with 1,2, 3, 4, etc. analyte molecules are not distinguished from each other(e.g., where ν=1, 2, 3, 4 are not differentiated from each other) andthe wells (e.g., locations, reaction vessels) are simply characterizedas “on” wells, then occurrences of ν=0 can by determined definitively asthe number of “off” wells. (P_(μ)(0)) may be calculated according toEquation 6:

$\begin{matrix}{{P_{\mu}(0)} = {{e^{- \mu}\left( \frac{\mu^{o}}{0!} \right)} = e^{- \mu}}} & \left( {{Eq}.\mspace{14mu} 6} \right)\end{matrix}$

and the number of expected occurrences, μ, may be determined based on arearrangement of Equation 5, as given in Equation 7:

μ=−ln[P _(μ)(0)]  (Eq. 7).

The number of occurrences of capture objects associated with no analytemolecules (or binding ligands), P_(μ)(0), is equal to 1 minus the totalnumber of capture objects with all other occurrences (e.g., captureobjects associated at least one analyte molecule or binding ligand) thenμ is given by Equation 8:

$\begin{matrix}{\mu = {\frac{{Number}\mspace{14mu} {of}\mspace{14mu} {Analyte}\mspace{14mu} {molecules}}{{Number}\mspace{14mu} {of}\mspace{14mu} {Capture}\mspace{14mu} {objects}} = {- {{\ln\left( {1 - {{fraction}\mspace{14mu} {of}\mspace{14mu} {``{on}"}\mspace{14mu} {wells}}} \right)}.}}}} & \left( {{Eq}.\mspace{14mu} 8} \right)\end{matrix}$

Rearranging Equation 8, the total number of analyte molecules in thefluid sample contained in the locations interrogated containing acapture object can be determined using Equation (9):

Number of Analyte molecules=−ln(1−fraction of “on” wells)×Number ofCapture objects   (Eq. 9).

Therefore, the total number of molecules can be determined from thefraction of “on” wells for a given number of wells containing captureobjects, and a measure of the concentration of analyte molecules in thefluid sample may be based at least in part on this number (as well as,e.g., any dilutions of the sample during the assay, the number andvolume of the wells containing capture objects interrogated, etc.). Thenumber of capture objects with 1, 2, 3, 4 etc. associated analytemolecules can also be determined by calculating P_(μ)(1), P_(μ)(2),P_(μ)(3) etc. from the μ determined and Equation 5.

As a non-limiting example of use of a Poisson distribution adjustment,in an assay where 26% of 50,000 capture objects interrogated were “on”(i.e., contained one or more analyte molecules and/or binding ligands)then the total number of analyte molecules present is calculated as−ln(1−0.26)×50,000=15,056 molecules. Of these 15,056 molecules, usingμ=−ln(1−0.26)=0.3011 in the Eq. 5 for ν=1, 11,141 capture objects arecalculated to have 1 analyte molecule, 1,677 capture objects 2 analytemolecules, 168 capture objects 3 analyte molecules, 13 capture objects 4analyte molecules, and 1 capture object 5 analyte molecules. A similaranalysis may be applied to embodiments where a statistically significantfraction of the spatially separated capture objects are associated withat least one binding ligand and a statistically significant fraction ofspatially separated capture objects are not associated with any bindingligands.

In some embodiments, wherein the ratio of locations comprising a captureobject associated with at least one analyte molecule and/or a bindingligand to locations containing a capture object free of any analytemolecule/binding ligand is high (e.g., greater than about 1:2, greaterthan about 1:1, greater than about 2:1, greater than about 4:1, greaterthan about 8:1, or greater), the determination of the concentration ofanalyte molecules in the fluid sample may be based at least in part onan intensity read-out determination. In such an embodiment, the totalintensity of the array (e.g., total fluorescence) may be determined anda measure of the concentration of analyte molecules in the fluid sampleis based at least in part on this determination.

In some embodiments, a measure of the concentration of analyte moleculesor particles in the fluid sample may be determined at least in part bycomparison of a measured parameter to a calibration standard. In somecases, a calibration curve may be used, similar to as described herein,wherein the total intensity is determined for a plurality of samplescomprising the analyte molecule at a known concentration using asubstantially similar assay format. For example, the number and/orfraction of locations that comprise a capture object associated with ananalyte molecule (e.g., based on a binary read-out), or alternatively,the total intensity of the array, may be compared to a calibration curveto determine a measure of the concentration of the analyte molecule inthe fluid sample. The calibration curve may be produced by completingthe assay with a plurality of standardized samples of knownconcentration under similar conditions used to analyze test samples withunknown concentrations. A calibration curve may relate the fraction ofthe capture objects determined to be associated with an analyte moleculeand/or binding ligand with a known concentration of the analytemolecule. The assay may then be completed on a sample containing theanalyte molecule in an unknown concentration, and number/fraction ofcapture objects determined to be associated with an analyte moleculeand/or binding ligand may be compared to the calibration curve, (or amathematical equation fitting same) to determine a measure of theconcentration of the analyte molecule in the fluid sample.

In one exemplary embodiment for performing a calibration, fourstandardized fluid samples comprising an analyte molecule in varyingconcentration (w, x, y, and z) are used. An assay (e.g., immobilizingthe analyte molecules with respect to a plurality of capture objects,optionally exposing the capture objects to at least one type of bindingligand, partitioning at least a portion of the capture objects into aplurality of discrete, separately addressable locations, detecting atleast a portion of the capture objects, etc.) is carried out for eachcalibration sample, and the number/fraction of capture objectscomprising an analyte molecule and/or binding ligand (b, c, d, and e) isdetermined. A plot/equation/look-up table, etc. is produced relating thevalues b, c, d, and e to concentrations w, x, y, and z, respectively, asdepicted in FIG. 12. The assay may be then be carried out undersubstantially identical conditions on a fluid sample containing ananalyte molecule of unknown concentration, wherein the resulting valueof number/fraction of capture objects comprising an analyte moleculeand/or binding ligand determined to detection of the capture objects isf. This value (f) may be plotted on the graph and a measure of theunknown concentration of the target analyte in the fluid sample (t) maybe determined. In some cases, the calibration curve may have a limit ofdetection, wherein the limit of detection is the lowest concentration ofanalyte molecules in a fluid sample that may be accurately determined.In some cases, the r² value of the calibration curve may be greater thanabout 0.5, greater than about 0.75, greater than about 0.8, greater thanabout 0.9, greater than about 0.95, greater than about 0.97, greaterthan about 0.98, greater than about 0.99, greater than about 0.9999, orabout 1. Values b, c, d, and e may be based on the absolute number ofmeasured locations/capture objects associated with an analyte molecule(or binding ligand), or a ratio of the number of locations containing acapture object associated with an analyte molecule (or binding ligand)to the number of locations containing a capture object not associatedwith any analyte molecules or a ratio of the number of locationscontaining a capture object associated with an analyte molecule (orbinding ligand) to the number of locations containing a capture objector a ratio of the number of locations containing a capture objectassociated with an analyte molecule (or binding ligand) to the totalnumber of locations addressed, etc. Any number of calibration standardsmay be used to develop the calibration curve (e.g., about 2, 3, 4, 5, 6,7, 8, 9, 10, or more, calibration standards).

In some embodiments, the concentration of analyte molecules in the fluidsample may be determined through use of a calibration curve using anassay system employing a computer. The computer may execute softwarethat may use the data collected to produce the calibration curve and/orto determine a measure of the concentration of analyte molecules in atest fluid sample from such calibration curve. For example, afluorescence image of an array comprising the plurality of captureobjects partitioned across the array may be collected and analyzed usingimage analysis software (e.g., IP Lab, BD Biosciences). The analysissoftware may automatically calculate the number of locations that havefluorescence intensity over the background intensity (e.g., a numberthat correlates to the number of locations which comprise an analytemolecule). The number of locations which comprise fluorescence intensityover the background intensity may be divided by the total number oflocations addressed, for example, to determine the fraction of locationswhich comprise an analyte molecule. The active location fraction may becompared to a calibration curve to determine a measure of theconcentration of analyte molecules in the fluid sample.

In certain embodiments, it may be possible to increase both the dynamicrange and the sensitivity of the assay by expanding the number oflocations into which the capture objects are partitioned and/or byadjusting the ratio of capture objects (e.g. beads) to analyte moleculesin the initial capture step. In certain cases, decreasing or increasingthe analyte-to bead ratio may result in more dynamic range. In somecases, as the volume of a sample increases, detecting small numbers ofanalyte molecule with accuracy, may, in some cases, become morechallenging for example, due to limitations of equipment, timeconstraints, etc. For example, to achieve the same efficiencies inlarger volume sample (e.g., 1 mL, 10 mL) as achieved with a smallervolume sample (e.g., 100 μL), more beads (e.g., 10 and 100 times morebeads, respectively) may be necessary, and thus, the beads may need tobe spatially segregated into larger number of locations, wherein thelarger number of locations may require an increased imaging area.

For the capture step, the choice of bead concentration may depend onseveral competing factors. For example, it can be advantageous ifsufficient beads are present to capture most of the target analyte fromthermodynamic and kinetic perspectives. As an exemplary illustration,thermodynamically, 200,000 beads in 100 μL that each have about 80,000capture components (e.g. antibodies) bound to correlates to an antibodyconcentration of about 0.3 nM, and the antibody-protein equilibrium atthat concentration may give rise to a relatively high capture efficiencyof target analyte molecules in certain cases (e.g. >70%). Kinetically,for 200,000 beads dispersed in 100 μL, the average distance betweenbeads can be estimated to be about 80 μm. Proteins the size of TNF-α andPSA (17.3 and 30 kDa, respectively), as exemplary analyte molecules, forexample, will typically tend to diffuse 80 μm in less than 1 min, suchthat, over a 2 hour incubation, capture of such analyte molecules willtend not to be limited kinetically. In addition, it can also beadvantageous to provide sufficient beads loaded onto the arrays to limitPoisson noise to a desired or acceptable amount. Considering as anexample a situation where 200,000 beads in a in 10 μL volume are loadedonto an array, typically about 20,000-30,000 beads may become trapped infemtoliter sized wells of the array. For a typical background signal(e.g. due to non specific binding, etc.) of 1% active beads, thisloading would be expected to result in a background signal of 200-300active beads detected, corresponding to a coefficient of variation (CV)from Poisson noise of 6-7%, which may be acceptable in typicalembodiments. However, bead concentrations above certain concentrationsmay be undesirable in certain cases in that they may lead to: a)increases in non-specific binding that may reduce signal-to-background;and/or b) undesirably low ratios of analyte-to-bead such that thefraction of active beads is too low, resulting in high CVs from Poissonnoise. In certain embodiments, considering a balance of factors such asthose discussed above, providing about 200,000 to 1,000,000 beads per100 μL of test sample may be desirable or, in certain cases optimal, forperforming certain assays of the invention.

For embodiments of the inventive assay employing one or more bindingligand(s) to label the captured analyte molecules, it may beadvantageous to, in certain instances, adjust the concentrations used toyield desirable or optimal performance. For example, considering anembodiment involving an analyte molecule that is a protein (capturedprotein) and employing a first binding ligand comprising a detectionantibody and a second binding ligand comprising an enzyme conjugate(e.g. SβG), the concentrations of detection antibody and enzymeconjugate (SβG) used to label the captured protein may in some cases belimited or minimized to yield an acceptable background signal (e.g. 1%or less) and Poisson noise. The choice of the concentrations ofdetection antibody and enzyme conjugate (SβG) used to label the capturedprotein can be factors in improving the performance of or optimizingcertain of the inventive assay methods. In certain cases, it may bedesirable for only a fraction of the capture proteins to be labeled soas to avoid saturating signals produced by the assay. For example, for aparticular assay where background levels observed are equivalent to ˜1-2fM of target protein, such that the ratio of analyte to bead may beabout 0.3-0.6, the number of active beads may be in the range of about25-40% if every protein was labeled with an enzyme, which may be higherthan desirable in some cases. To produce background signals that may becloser to a lower end of the dynamic range for a digital detectionassay—considering e.g. that in certain cases 1% active beads may providea reasonable noise floor for background in digital detection assays ofthe invention—appropriate labeling of the captured protein canpotentially be achieved by kinetic control of the labeling steps, eitherby limiting or minimizing the concentrations of both labeling reagentsor by using shorter incubation times. For example, in an embodimentwhere label concentrations are minimized, use of a standard ELISAincubation time may provide acceptable results; e.g. using a total assaytime of ˜6 h. This length of time may be acceptable for testing thattolerates a daily turnaround time for samples. For shorter turnaroundtimes of, for example, <1 hour (e.g., for point-of-care applications),the assay could be performed with shorter incubations with higherconcentrations of labels.

In some embodiments, accuracy of a particular method of determiningconcentration with the inventive assays may be compromised, i.e. bothabove and below the thresholds of the dynamic range for the particularmethod. For example, as the concentration of the capture objectsassociated with an analyte molecule goes down, eventually, when belowthe lower limit of the dynamic range, the number of capture objectsassociated an analyte molecule may be too low to observe a sufficientnumber of occupied locations to obtain a reliable and reproduciblemeasurement. In such a situation, the number of locations could bedecreased in order to make sure that at least some (e.g., astatistically significant number) of them are occupied by a captureobject associated with an analyte molecule, and/or the sample testedcould be concentrated and/or the number of capture objects incubatedwith the sample could be decreased, etc. to increase the number/fractionof positive capture objects detected. On the other hand, a binaryread-out system/method may be above its upper threshold of accuracyand/or utility when, for example, loading approaches saturation with“on” capture objects such that substantially 100% of the locationscontain at least one capture object associated with an analyte molecule.At this limit, discrimination between two samples with concentrationsfalling in this range may not be feasible using a binary read-outsystem/method. In such a situation, to provide a more accurate result, agreater number of locations could be used, the concentration of thesample could be reduced, for example, through serial dilutions, thenumber of capture objects incubated with the sample could be increased,etc. to decrease the number/fraction of positive capture objectsdetected, and/or an intensity read-out system/method could be employed.

In some embodiments, the concentration of analyte molecules or particlesin the fluid sample that may be substantially accurately determined isless than about 5000 fM, less than about 3000 fM, less than about 2000fM, less than about 1000 fM, less than about 500 fM, less than about 300fM, less than about 200 fM, less than about 100 fM, less than about 50fM, less than about 25 fM, less than about 10 fM, less than about 5 fM,less than about 2 fM, less than about 1 fM, less than about 500 aM(attomolar), less than about 100 aM, less than about 10 aM, less thanabout 5 aM, less than about 1 aM, less than about 0.1 aM, less thanabout 500 zM (zeptomolar), less than about 100 zM, less than about 10zM, less than about 5 zM, less than about 1 zM, less than about 0.1 zM,or less. In some cases, the limit of detection (e.g., the lowestconcentration of an analyte molecule which may be determined in solutionsubstantially accurately) is about 100 fM, about 50 fM, about 25 fM,about 10 fM, about 5 fM, about 2 fM, about 1 fM, about 500 aM(attomolar), about 100 aM, about 50 aM, about 10 aM, about 5 aM, about 1aM, about 0.1 aM, about 500 zM (zeptomolar), about 100 zM, about 50 zM,about 10 zM, about 5 zM, about 1 zM, about 0.1 zM, or less. In someembodiments, the concentration of analyte molecules or particles in thefluid sample that may be substantially accurately determined is betweenabout 5000 fM and about 0.1 fM, between about 3000 fM and about 0.1 fM,between about 1000 fM and about 0.1 fM, between about 1000 fM and about0.1 zM, between about 100 fM and about 1 zM, between about 100 aM andabout 0.1 zM. The concentration of analyte molecules or particles in afluid sample may be considered to be substantially accurately determinedif the measured concentration of the analyte molecules or particles inthe fluid sample is within about 10% of the actual (e.g., true)concentration of the analyte molecules or particles in the fluid sample.In certain embodiments, the measured concentration of the analytemolecules or particles in the fluid sample may be within about 5%,within about 4%, within about 3%, within about 2%, within about 1%,within about 0.5%, within about 0.4%, within about 0.3%, within about0.2% or within about 0.1%, of the actual concentration of the analytemolecules or particles in the fluid sample. In some cases, the measureof the concentration determined differs from the true (e.g., actual)concentration by no greater than about 20%, no greater than about 15%,no greater than 10%, no greater than 5%, no greater than 4%, no greaterthan 3%, no greater than 2%, no greater than 1%, or no greater than0.5%. The accuracy of the assay method may be determined, in someembodiments, by determining the concentration of analyte molecules in afluid sample of a known concentration using the selected assay method.

In some embodiments, more than one of the above described types ofanalysis and quantification methods may be employed with the same systemand in a single assay. For example, in embodiments where the analytemolecules are present at lower concentration ranges, single analytemolecules can be detected, and the data may be analyzed using a digitalanalysis method (binary quantification). In some cases using binaryquantification, as previously described, the data may be processed usinga Poisson distribution adjustment. At higher concentration ranges (e.g.,where it may become challenging or inaccurate to perform binaryquantification), the data may be analyzed using an analog analysismethod, based, for example on measured relative signal intensities(intensity read-out determination). In certain embodiments, the resultsof the two analysis methods (digital and analog) may both be utilized bya single assay system/protocol of the invention by linking the twomethods using a single calibration curve. For example, in someembodiments, at low concentration levels (e.g., in the digital/binaryconcentration range), a measure of the concentration of analytemolecules in a fluid sample may be determined at least in part bycounting beads as either “on” (e.g., by determining if the reactionvessel contains a bead associated with an analyte molecule) or “off”(e.g., by determining if the reaction vessel contains a bead notassociated with any analyte molecule). At low ratios of analytemolecules to beads (e.g., less than about 1:5, less than about 1:10,less than about 1:20, etc.), substantially all of the beads areassociated with either zero or a single analyte molecule. In this range,the percentage of active beads (e.g., “on” reaction vessels) increaseslinearly with increasing analyte concentration, and a digital analysismethod may be most suitable.

As the analyte concentration increases, however, more of the beads willassociate with more than one analyte molecule. Therefore, as the analyteconcentration increases (but still in the digital range), the percentageof active beads in a population generally will not be linearly relatedto the bulk analyte concentration as some of the beads may associatewith more than one analyte molecule. In these concentration ranges, thedata may be advantageously analyzed using a digital analysis method withthe above described Poisson distribution adjustment. The above describednon-linear effect can be accounted for using Poisson distributionadjustment across substantially the concentration range in which thereremains a statistically significant fraction of beads not associatedwith any analyte molecules or particles in the sample. For example,ranges of percentage of active beads (i.e. “on” beads divided by totalbeads multiplied by 100%) for which a digital analysis method may beable to accurately determine a measure of the concentration, include upto about 20% active beads, up to about 30% active beads, up to about 35%active beads, up to about 40% active beads, up to about 45% activebeads, up to about 50% active beads, or more. In many cases whenoperating in the ranges above, using a Poisson distribution adjustmentwill improve accuracy.

Above a certain active bead percentage (i.e., where there is no longer astatistically significant fraction of beads present in the populationthat are not associated with any of analyte molecules or particles, or,potentially advantageously for situations where there may be astatistically significant fraction of beads present in the populationthat are not associated with any of analyte molecules or particles butthat result in active bead percentages above a certain level—e.g.,greater than or substantially greater than about 40%) (or activelocation percentage, in embodiments where beads are not employed))improvements in accuracy and/or reliability in the determination ofanalyte molecule concentration may potentially be realized by employingan intensity measurement based analog determination and analysis ratherthan or supplementary to a digital/binary counting/Poisson distributionadjustment as previously described. At higher active bead percentages,the probability of an active bead (e.g., positive reaction vessel) beingsurrounded by other active beads (e.g., positive reaction vessels) ishigher and may in certain assay set ups create certain practicalchallenges to exclusively using the digital/binary determination method.For example, in certain embodiments, leakage of a detectable componentinto a reaction vessel from an adjacent reaction vessel may occur tosome extent. Use of an analog, intensity level based technique in suchsituations can potentially yield more favorable performance. In anintensity measurement based analog determination and analysis, theassociation of multiple analyte molecules at relatively highconcentrations with single beads is quantified. The intensity of atleast one signal from the plurality of reaction vessels which contain atleast one analyte molecule may be determined. In some cases, theintensity is determined as the total overall intensity for the reactionvessels containing at least one analyte molecule (e.g., the intensity ofthe reaction vessels in determined as a whole). In other cases, theintensity of each reaction vessel with a signal may be determined andaveraged, giving rise to an average bead signal (ABS).

According to certain embodiments, an inventive assay system may includea link between the results/parameters of the two analysismethods/systems (i.e. digital and analog), for example, with the aid ofa calibration curve, so that the system is able to operate in multiplemodes of quantification depending on the signal relating to thenumber/fraction of “on” beads detected. Such systems can havesubstantially expanded dynamic ranges in certain cases. Furtherdescription of such systems which can combine and use more than onequantification method for a single assay is provided in commonly ownedU.S. patent application Ser. No. ______ (not yet determined), entitled“Methods and systems for extending dynamic range in assays for thedetection of molecules or particles” by Rissin et al., filed Mar. 24,2010 (Attorney Docket No. Q0052.70013US01), incorporated by reference.

The following examples are included to demonstrate various features ofthe invention. Those of ordinary skill in the art should, in light ofthe present disclosure, will appreciate that many changes can be made inthe specific embodiments which are disclosed while still obtaining alike or similar result without departing from the scope of the inventionas defined by the appended claims. Accordingly, the following examplesare intended only to illustrate certain features of the presentinvention, but do not necessarily exemplify the full scope of theinvention.

EXAMPLE 1

This following example describes materials used in Examples 2-19.Optical fiber bundles were purchased from Schott North America(Southbridge, Mass.). Non-reinforced gloss silicone sheeting wasobtained from Specialty Manufacturing (Saginaw, Mich.). Hydrochloricacid, anhydrous ethanol, and molecular biology grade Tween-20 werepurchased from Sigma-Aldrich (Saint Louis, Mo.). 2.8-um(micrometer)-diameter tosyl-activated magnetic beads were purchased fromInvitrogen (Carlsbad, Calif.). 2.7-um-diameter carboxy-terminatedmagnetic beads were purchased from Varian, Inc. (Lake Forest, Calif.).Monoclonal anti-human TNF-α capture antibody, polyclonal anti-humanTNF-α detection antibody, and recombinant human TNF-α were purchasedfrom R&D systems (Minneapolis, Minn.). Monoclonal anti-PSA captureantibody and monoclonal detection antibody were purchased fromBiosPacific (Emeryville, Calif.); the detection antibody wasbiotinylated using standard methods. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (NHS), andSuperBlock® T-20 Blocking Buffer were purchased from Thermo Scientific(Rockford, Ill.). DNA was purchased from Integrated DNA Technologies(Coralville, Iowa) and/or purified DNA was ordered from Integrated DNATechnologies (Coralville, Iowa). Streptavidin-β-galactosidase (SβG) waspurchased from Invitrogen or conjugated in house using standardprotocols. Resorufin-β-D-galactopyranoside (RGP) was purchased fromInvitrogen (Carlsbad, Calif.). The fiber polisher and polishingconsumables were purchased from Allied High Tech Products (RanchoDominguez, Calif.).

EXAMPLE 2

The following describes a non-limiting example of the preparation of2.8-um-diameter magnetic beads functionalized with protein captureantibody. 600 uL (microliter) of 2.8-um-diameter tosyl-activatedmagnetic bead stock (1.2×10⁹ beads) was washed three times in 0.1 Msodium borate coating buffer pH 9.5. 1000 ug (microgram) of captureantibody was dissolved in 600 uL of sodium borate coating buffer. 300 uLof 3M ammonium sulfate was added to the antibody solution. The 600 uL ofbead solution was pelleted using a magnetic separator and thesupernatant was removed. The antibody solution was added to the beadsand the solution was allowed to mix at 37° C. for 24 hours. Afterincubation, the supernatant was removed and 1000 uL of PBS buffercontaining 0.5% bovine serum and 0.05% Tween-20 was added to the beads.The beads were blocked overnight (˜8 hours) at 37° C. The functionalizedand blocked beads were washed three times with 1 ml PBS buffercontaining 0.1% bovine serum and 0.05% Tween-20. Finally, 1 mL of PBScontaining 0.1% bovine serum, 0.05% Tween-20, and 0.02% sodium azide wasadded to the functionalized and blocked beads. 50 uL aliquots werestored at 4° C. for later use.

EXAMPLE 3

The following describes a non-limiting example of the preparation of2.7-um-diameter magnetic beads functionalized with protein captureantibody. 500 uL of 2.7-um-diameter carboxy-terminated magnetic beadsstock (1.15×10⁹ beads) was washed twice in 0.01 M sodium hydroxide,followed by three washes in deionized water. Following the final wash,the bead solution was pelleted and the wash solution was removed. 500 uLof a freshly prepared 50 mg/mL solution of NHS in 25 mM MES, pH 6.0, wasadded to the bead pellet and mixed. Immediately, a 500 uL of a freshlyprepared 50 mg/mL solution of EDC in 25 mM MES, pH 6.0, was added to thebead solution and mixed. The solution was then allowed to mix for 30 minat room temperature. After activation, the beads were washed twice with25 mM MES at pH 5.0. Meanwhile, 1000 uL of 25 mM MES at pH 5.0 was usedto dissolve 1000 ug of capture antibody. The antibody solution was thenadded to the activated beads and the coupling reaction was allowed toproceed for 3 hours at room temperature. After incubation, thesupernatant was removed using the magnetic separator, and 1000 uL of 100mM Tris-HCl (pH 7.4) was added and allowed to mix at room temperaturefor one hour to block any remaining reactive sites. Finally, thefunctionalized beads were stored in 1 mL of SuperBlock blocking buffer,and 0.02% sodium azide was added to the functionalized and blockedbeads. 50 uL aliquots were stored at 4° C. for later use.

EXAMPLE 4

The following describes a non-limiting example of the preparation of2.7-um-diameter magnetic beads functionalized with DNA. 120 μL of2.7-um-diameter carboxy-terminated magnetic beads was washed three timeswith 0.01 M NaOH, followed by deionized water for another three times.500 uL of freshly prepared 50 mg/mL NHS in cold 25 mM MES (pH 6) wasadded to the pellet of beads after the final wash, and the beads werere-suspended by vortexing briefly. 500 uL of freshly prepared 50 mg/mLEDC solution in cold 25 mM MES (pH 6) was immediately added to this beadsuspension and mixed for 30 min. After activation, the beads were washedthree times with cold 25 mM MES (pH 5). DNA capture probe with aminemodification at 5′ end (5′-NH2/C12-GTT GTC AAG ATG CTA CCG TTC AGA G-3′(SEQ ID NO. 1)) was dissolved in nuclease-free water to make a 2.6 mMstock solution. 60 μL of the DNA stock was added to 600 uL of thecoupling buffer that contains 0.1 M sodium phosphate and 0.5 M NaCl, pH8. The resulting DNA solution was added to the washed beads and mixedfor 3 hours at room temperature. The bead suspension was vortexed every30 min during the reaction. After incubation, the DNA supernatant wasremoved and 1 mL of 100 mM Tris-HCL (pH 7.4) was added to the pellet andmixed for 1 hour to inactivate the remaining binding sites on the beads.Finally, the beads were washed in Tris-EDTA (TE) buffer and 0.05%Tween-20 for three times, and stored in TE buffer containing 0.05%Tween-20 and 0.02% sodium azide at 4° C.

EXAMPLE 5

The following describes a non-limiting example of the capture ofproteins on magnetic beads and formation of enzyme-labeledimmunocomplex. Test solutions containing the protein of interest wereincubated with suspensions of magnetic beads functionalized with captureantibody (e.g., see Example 2) for 1 h at 37° C. The beads were thenseparated and washed three times in PBS. The beads were resuspended andincubated with solutions containing detection antibodies for 30 min at37° C. The beads were then separated and washed three times in PBS. Thebeads were incubated with solutions containing SβG (e.g., targetanalyte) for 30 min at 37° C., separated, and washed six times in PBSand 0.1% Tween-20. The beads were then resuspended in 10 uL of PBS inorder to load into the wells of the fiber bundle arrays.

EXAMPLE 6

The following describes a non-limiting example of the capture of DNA onmagnetic beads and formation of enzyme-labeled complex (FIG. 25). Beadsfunctionalized with DNA capture probe (e.g., see Example 4) that isspecific to the complementary target DNA of interest were incubated withsolutions containing the target DNA (5′-TT GAC GGC GAA GAC CTG GAT GTATTG CTC C TCT GAA CGG TAG CAT CTT GAC AAC-3′ (SEQ ID NO. 2)) (e.g.,target analyte) for 2 hrs. After incubation, the DNA target solution wasremoved and the beads were washed three times in 0.2×SSC buffercontaining 0.1% Tween-20. The beads were then resuspended and mixed with10 nM biotinylated signal probe (5′-TAC ATC CAG GTC TTC GCC GTCAA/Biotin/-3′ (SEQ ID NO. 3)) (e.g., first type of binding ligand) thatis also specific to the target DNA for 1 hr. The beads were then washedthree times in 0.2×SSC buffer containing 0.1% Tween-20 after removingthe signal probe. A solution 10 pM containing SβG (e.g., second type ofbinding ligand comprising an enzymatic component) was then added to thebead pellet, resuspended, and mixed for 1 hr. The beads were thenseparated and washed six times in 5× PBS buffer containing 0.1%Tween-20. The beads were then resuspended in 10 μL of PBS and loadedonto a femtoliter well array.

EXAMPLE 7

The following example describes the capture of biotin-labeled DNA onmagnetic beads and formation of enzyme-labeled complex, according to anon-limiting embodiment (see FIG. 24). Beads functionalized with DNAcapture probe that is specific to DNA of interest were incubated with 1uM target DNA-biotin (5′-biotin-C TCT GAA CGG TAG CAT CTT GAC AAC-3′(SEQ ID NO. 4)) overnight (16 hrs) in TE buffer containing 0.5M NaCl and0.01% Tween-20. After incubation, the DNA target solution was removedand the beads were washed three times in PBS buffer containing 0.1%Tween-20. The bead stock was distributed into a microtiter plate giving400,000 beads per well in 100 uL. The buffer was aspirated from themicrotiter plate wells, the beads were resuspended and incubated withvarious concentrations of SβG in Superblock containing 0.05% Tween-20for 5 hr. In some cases, the beads were resuspended every 30 min duringthe incubation. The beads were then separated and washed six times with5× PBS buffer containing 0.1% Tween-20. Finally, the beads wereresuspended in 10 uL of PBS containing 0.1% Tween-20. In someembodiments, the beads were then separated and washed six times with 5xPBS buffer containing 0.1% Tween-20. For detection of enzyme, the beadswere either: a) resuspended in 20 μL of PBS containing 0.1% Tween-20,and 10 μL aliquots were loaded onto two femtoliter well arrays fordetection, or; b) resuspended in 100 μL of 100 μM RGP in PBS, incubatedfor 1 h at room temperature, and read on a fluorescence plate reader(Infinite M200, Tecan).

EXAMPLE 8

The following describes a non-limiting example of the preparation ofmicrowells arrays. Optical fiber bundles approximately 5-cm long weresequentially polished on a polishing machine using 30-, 9-, and1-micron-sized diamond lapping films. The polished fiber bundles werechemically etched in a 0.025 M HCl solution for 130 seconds, and thenimmediately submerged into water to quench the reaction. To removeimpurities from etching, the etched fibers were sonicated for 5 s andwashed in water for 5 min. The fibers were then dried under vacuum andexposed to air plasma for 5 min to clean and activate the glass surface.The arrays were silanized for 30 minutes in a 2% solution of silane tomake the surfaces hydrophobic.

EXAMPLE 9

The following describes a non-limiting example of the loading of beadsinto microwells. To apply the solution of beads to the etched wells in afiber bundle, clear PVC tubing ( 1/16″ I.D. ⅛″ O.D.) and clear heatshrink ( 3/16″ ID) were cut into approximately 1 cm long. A piece of PVCtubing was first put onto the etched and functionalized end of a fiberbundle to create a reservoir to hold the bead solution, followed by theapplication of heat shrink around the interface between the PVC tubingand fiber bundle to provide a tight seal. 10 uL of the concentrated beadsolution was pipetted into the reservoir created by the PVC tubing. Thefiber bundle was then centrifuged at 3000 rpm (1333 g) for 10 minutes toforce the beads into the etched wells. The PVC tubing/heat shrinkassembly was removed after centrifugation. The distal end of the fiberbundle was dipped in PBS solution to wash off excess bead solution,followed by swabbing the surface with deionized water.

EXAMPLE 10

The following describes a non-limiting example of the detection of beadsand enzyme-labeled beads in microwell arrays. A custom-built imagingsystem containing a mercury light source, filter cubes, objectives, anda CCD camera was used for acquiring fluorescence images. Fiber bundlearrays were mounted on the microscope stage using a custom fixture. Adroplet of β-galactosidase substrate (RPG) was placed on the siliconegasket material, and put into contact with the distal end of the fiberarray. The precision mechanical platform moved the silicone sheet intocontact with the distal end of the etched optical fiber array, creatingan array of isolated femtoliter reaction vessels. Fluorescence imageswere acquired at 577 nm with an exposure time 1011 ms. Five frames (at30 seconds per frame) were taken for each fiber bundle array. Thefluorescent images were analyzed using image analysis software todetermine the presence or absence of enzymatic activity within each wellof the microwell array. The data was analyzed using a developed imageprocessing software using MathWorks MATLAB and MathWorks ImageProcessing toolbox. The software aligns acquired image frames,identifies reaction vessel positions, locates reaction vessels withbeads and measures the change in reaction vessel intensity over apredefined time period. Reaction vessels containing beads withsufficient intensity growth over all data frames are counted and thefinal number of active reaction vessels is reported as a percentage ofall identified reaction vessels.

As well as fluorescence, the arrays were imaged with white light toidentify those wells that contain beads. After acquiring thefluorescence images, the distal (sealed) end of the fiber bundle arrayswere illuminated with white light and imaged on the CCD camera. Due toscattering of light by the beads, those wells that contained a beadappeared brighter in the image than wells without beads. Beaded wellswere identified using this method by software.

EXAMPLE 11

The following describes a non-limiting example of the loading of beadsinto an array of microwells (FIG. 13). Arrays of 50,000 microwells wereprepared as described above. 2.8 um beads were prepared as describedabove. 10-uL solutions containing different numbers of beads (from80,000 to 2 million beads) were prepared as described above. Beads wereloaded into the arrays of microwells as described above. The arrayloaded with a solution comprising 2 million beads was imaged usingscanning electron microscopy (SEM). SEM showed that >99% of the 50,000wells contained a bead, and each of these well only contained a singlebead. The arrays loaded with 80,000 to 200,000 beads were imaged usingwhite-light microscopy and image analysis was used to identify wellsthat contained a bead. The number of beads per array was determined overthree arrays and plotted as a function of number of beads in solution(FIG. 13B). From FIG. 13B, in this embodiment, the number of beadsloaded is a fraction of those provided in solution and not every wellcontains a bead at these assay-relevant bead loading concentrations. Insome cases, the presence of a bead in a well (using white light images)may be correlated to those wells that contain enzymatic activity. Insuch cases, the read-out may be ratiometric (% active beads) andnormalized for variation in bead loading.

EXAMPLE 12

The following describes a non-limiting example of bead filling as afunction of well depth (FIG. 14). In some embodiments, a single beadcontaining single analyte molecules can be delivered into a microwell sothat they can be spatially isolated and sealed. To achieve thissituation, the well depth and width may be carefully controlled toparameters optimized for a given bead diameter. FIGS. 14A-14C show SEMimages of beads loaded as described above into arrays of microwellswhere the well depth was controlled by etching for different times. Onaverage, the wells etch at a rate of approximately 1.5 to 1.7 μm perminute. Therefore, wells of 3.25 um depth are produced in about 115 to130 s. For a well depth of 2.5 um (FIG. 14A), very few beads areretained in the microwells as they are too shallow and detection ofsingle analytes may be poor. At 3-um depth (FIG. 14B), SEM images showgood filling of single beads into single wells, and a low occurrence oftwo beads in one well; this array may seal well and allow large numbersof single beads to be interrogated for the presence of a single analyte.For 3.5-um deep wells (FIG. 14C), many of the wells contain two beads aswell as those that contain one. The presence of a second bead above theplane of the array may deteriorate the sealing of the array as describedabove and may denigrate the quality of single bead isolation. Theseexperiments indicated that an optimal well depth for 2.8-um diameterbeads, in this embodiment, is between about 3 and about 3.25 um. Whilethis range is optimal, it is also possible to perform the inventivemeasurements using well depths of 3.6 um, i.e., the upper limit asindicated by Eq.(4).

EXAMPLE 13

The following example describes the comparison of a non-limiting methodof the present invention versus a conventional plate reader fordetecting enzyme (see FIGS. 15A, 15B, and 16). Biotin-DNA beads wereprepared as described above. These beads were then incubated with a lowconcentration of S βG such that beads statistically contained eitherzero or one enzymes. These beads were loaded into microwells, sealed,and imaged as described above; FIGS. 15A and 15B show representativeimages. In some cases, an increase in sensitivity to enzyme label thatcomes from isolating single beads is observed as compared to traditionalbulk measurements. Beads coated with DNA were prepared, incubated withbiotinylated DNA, and then incubated with various concentrations of SβG(from 350 aM to 320 fM) as described above. Enzymes on these beads werethen measured in two ways. First, the beads were loaded into microwellarrays, sealed and imaged as described above. The fraction of activewells was determined as described above and is plotted as a function ofthe concentration of SβG in FIGS. 16A, the lower range expanded in FIG.16B. Second, the beads were incubated with 100 uL of RPG in a microtiterplate for one hour and read on a fluorescence plate reader. Thefluorescence signal as a function of the concentration of SβG is plottedin FIG. 16C. The lower limit of detection (LOD) of the inventive method(defined as the concentration at which the signal rises above threestandard deviations over the background) in this experiment was 384 zM.The LOD of the bulk measurement on the plate reader was 14.5 fM. Thesingle molecule array approach of the present invention, therefore,provided an increase of 37,760-fold in sensitivity to enzyme label overthe plate reader. It should be noted that at the concentrations testedstatistically only zero or single analytes should be detected on beads;for example, the ratio of enzymes to beads at 350 aM was21,070/400,000=0.053.

EXAMPLE 14

The following example illustrates the precision of detection in a methodas described herein, in a non-limiting embodiment. Detection of singlemolecules may allow for high precision. In theory, the lowest variancein the measurement is the Poisson noise associated with counting smallnumbers of events. In this non-limiting example, the % Poisson Noise isgiven by √N/N, where N is the number of active (enzyme-associated)beads. FIG. 17 shows a plot of the % Poisson Noise against theexperimental variance over three measurements (% CV) from theexperimental data in FIG. 16B. As can be seen, the imprecision of themeasurement (% CV) tracks closely with the Poisson Noise, suggestingthat the Poisson noise may limit the precision of the methods in somecases.

EXAMPLE 15

The following non-limiting example describes the detection of PSA inserum (FIG. 18). 2.8-um-diameter beads coated in anti-PSA antibody wereprepared as described above. These beads were incubated with 25% bovineserum or 25% bovine serum spiked with 50 fM PSA. The beads were thenlabeled with anti-PSA detection antibody and three differentconcentrations of SβG (1, 10, or 100 pM). The beads were then loadedinto microwell arrays, sealed and imaged as described above. Imageanalysis was used to determine the fraction of beads that contained anenzyme. These data show that the invention can be used to detect lowconcentrations of proteins in serum by performing ELISAs on single beadsand detecting single enzyme labels. Because of the high efficiency ofthe capture of analyte using beads in this invention, the concentrationof enzyme label used can be varied to only label a fraction of theanalytes captured on the beads in order to optimize thesignal-to-background and dynamic range of the measurement. In this dataset, 1 pM of enzyme label gave an optimal signal-to-background ratio.

EXAMPLE 16

The following non-limiting example describes the detection of TNF-α(FIG. 19). 2.8-um-diameter beads coated in anti-TNF-α antibody wereprepared as described above. These beads were incubated with 25% bovineserum or 25% bovine serum spiked with 100 fM TNF-α. The beads were thenlabeled with anti-TNF-α detection antibody (e.g., first binding ligand)and two different concentrations of SβG (1 or 100 pM) (e.g., secondbinding ligand comprising an enzymatic component). The beads were thenloaded into microwell arrays, sealed and imaged as described above.Image analysis was used to determine the fraction of beads thatcontained an enzyme. These data show that the invention can be used todetect low concentrations of TNF-α in serum by performing ELISAs onsingle beads and detecting single enzyme labels. As in the previousexample, the amount of enzyme label can be varied to ensure that themeasurement detects only single enzyme labels on the beads and optimizethe signal-to-background ratio. In this particular example, thebackgrounds are very low so the signal-to-background ratio is optimal atan enzyme label concentration of 1 pM and 100,000 beads.

EXAMPLE 17

The following non-limiting example describes the detection of DNA inbuffer (FIG. 20). 2.7-um-diameter beads functionalized with a capturesequence of DNA were prepared as described as above. These beads werethen incubated with various concentrations of target DNA and thenlabeled with a biotinylated signal probe DNA sequence as describedabove. The beads were then labeled by incubating with variousconcentrations of SβG (1, 10, or 100 pM). The beads were then loadedinto microwell arrays, sealed and imaged as described above. Imageanalysis was used to determine the fraction of beads contained anenzyme. These data show that the invention can be used to detect lowconcentrations of DNA by forming sandwich-like complexes on single beadsand detecting single enzyme labels. As in the case of protein detection,the amount of enzyme label can be varied to ensure that there arestatistically one or zero enzymes per bead even in the case where thereare more than one target DNA molecules captured. This allows the dynamicrange and signal-to-background of the single molecule measurement to beoptimized. In this case, 10 pM of SβG gave the optimalsignal-to-background.

COMPARATIVE EXAMPLE 18

The following non-limiting example describes a method using detectioncomprising chemiluminscence from alkaline phosphatase (FIG. 21).Different concentrations of 10 uL of alkaline phosphatase were mixedwith 90 uL of a solution containing the most sensitive chemiluminescentsubstrate available (APS-5; Lumigen Inc.) in a microtiter plate andincubated for 5 mins. The microtiter plate was then read inchemiluminescence mode of a plate reader. FIG. 21 shows a plot ofchemiluminescence as a function of concentration of alkalinephosphatase. The lowest concentration of enzyme that could be detectedabove background was 100 aM and the calculated limit of detection was 50aM, close to the reported value of 30 aM. Single molecule detection ofβ-galactosidase on beads in this invention (LOD=220 zM) is, therefore,more than 100 times more sensitive than chemiluminscent detection ofalkaline phosphatase, the most sensitive enzyme label system that iscommercially available.

EXAMPLE 19

The clinical use of protein biomarkers for the differentiation ofhealthy and disease states, and for monitoring disease progression,requires the measurement of low concentrations of proteins in complexsamples. Certain current immunoassays can measure proteins atconcentrations above 10⁻¹² M, whereas the concentration of the majorityof proteins important in cancer, neurological disorders, and the earlystages of infection are thought to circulate in the range from 10⁻¹⁶ to10⁻¹² M. For example: a 1 mm³ tumor composed of a million cells thateach secrete 5000 proteins into 5 L of circulating blood translates to˜2×10⁻¹⁵ M (or 2 femtomolar, fM); early HIV infection with seracontaining 2-3000 virions equates to concentrations of p24 antigenranging from 60×10⁻¹⁸ M (60 attomolar, aM) to 15×10⁻¹⁵ M (15femtomolar). Attempts to develop protein-based detection methods capableof detecting these concentrations have focused on the replication ofnucleic acid labels on proteins, or on measuring the bulk, ensembleproperties of labeled protein molecules. Sensitive methods for detectingproteins have, however, lagged behind those for nucleic acids, such asthe polymerase chain reaction (PCR), limiting the number of proteins inthe proteome that have been detected in blood. The isolation anddetection of single protein molecules provides the most direct methodfor measuring extremely low concentrations of proteins, although thesensitive and precise detection of single protein molecules has provenchallenging. The following describes a non-limiting exemplary method fordetecting thousands of single protein molecules simultaneously using thesame reagents as the gold standard for detecting proteins, namely, theenzyme-linked immunosorbent assay (ELISA). The method can detectproteins in serum at attomolar concentrations and may enable themeasurement of a single molecule in blood.

The method makes use of arrays of femtoliter-sized reaction chambers(FIG. 23) that can isolate and detect single enzyme molecules. In thefirst step, a sandwich antibody complex is formed on microscopic beads,and the bound complexes are labeled with an enzyme reporter molecule, asin a conventional bead-based ELISA. When assaying samples containingextremely low concentrations of protein, the ratio of protein molecules(and the resulting enzyme label complex) to beads is small (typicallyless than 1:1) and, as such, the percentage of beads that contain alabeled immunocomplex follows a Poisson distribution, leading to singleimmunocomplexes on individual beads. For example, if 50 aM of a proteinin 0.1 mL (3000 molecules) was captured on 200,000 beads, then 1.5% ofthe beads would have one protein molecule and 98.5% would have zeroprotein molecules (FIG. 23B). It is typically not possible to detectthese low numbers of proteins using conventional detection technology(e.g., a plate reader), because the fluorophores generated by eachenzyme diffuse into a large assay volume (typically 0.1-1 mL), and ittakes hundreds of thousands of enzyme labels to generate a fluorescencesignal above background (FIG. 24A). The method of this Example enablesthe detection of very low concentrations of enzyme labels by confiningthe fluorophores generated by individual enzymes to extremely smallvolumes (˜50 fL), leading to a high local concentration of fluorescentproduct molecules. To achieve this localization in an immunoassay, inthe second step of the method the immunoassay beads are loaded into anarray of femtoliter-sized wells (FIG. 23B). The loaded array is thensealed against a rubber gasket in the presence of a droplet offluorogenic enzyme substrate, isolating each bead in a femtoliterreaction chamber. Beads possessing a single enzyme-labeled immunocomplexgenerate a locally high concentration of fluorescent product in the50-fL reaction chambers. By using standard fluorescence imaging on amicroscope, it is possible to detect single enzyme molecules, and toimage tens to hundreds of thousands of immunocomplexes substantiallysimultaneously. By isolating the enzymes associated with eachimmunocomplex, each complex can give rise to a high measurable signalwhich can result in substantially improved sensitivity over bulkmeasurements. The protein concentration in the test sample, in somecases, is determined by simply counting the number of wells containingboth a bead and fluorescent product relative to the total number ofwells containing beads (FIG. 23D). The concentration is then determineddigitally rather than by using the total analog signal.

FIG. 23A shows the capturing and labeling single protein molecules onbeads using standard ELISA reagents. FIG. 23B shows the loading of beadsinto femtoliter microwell arrays for isolation and detection of singlemolecules. FIG. 23C shows an SEM image of a small section of afemtoliter well array after bead loading. 2.7-μm-diameter beads wereloaded into an array of wells with diameters of 4.5 μm and depths of3.25 μm. FIG. 23D shows a fluorescence image of a small section of thefemtoliter well array after signals from single molecules are generated.While the majority of femtoliter chambers contain a bead from the assay,only a fraction of those beads possess catalytic enzyme activity,indicative of a single, bound protein. The concentration of protein inbulk solution may be correlated to the percentage or number of beadsthat have bound a protein molecule. The exemplary assay was capable ofproviding linearity over ˜4.5 logs for 50,000 beads.

FIG. 24 shows the digitization of enzyme-linked complexes can increasesensitivity substantially compared to bulk, ensemble measurements. FIG.24 shows a log-log plot of signal output (% active beads for assaycomprising the use of capture objects; Relative Fluorescence Units(r.f.u.) for plate reader) as a function of the concentration of SβG.SβG concentrations for the ensemble readout ranged from 3 fM to 300 fM,with a detection limit of 15×10⁻¹⁵ M (15 fM; line (i)). For theexemplary assay according to the current invention, SβG concentrationsranged from 350 zM to 7 fM, demonstrating a linear response over 4.5logs, with a detection limit of 220×10⁻²¹ M (220 zM; line (ii)). Errorbars are based on the standard deviation over three replicates for bothtechnologies. LODs were determined from the signal at three standarddeviations above background. Table 1 provides information regarding theimprecision of this exemplary assay according to the current inventionrelating to Poisson noise of counting single events. The intrinsicvariation (Poisson noise) of counting single active beads is given byfin. Comparing the Poisson noise associated coefficient of variation (%CV) with the % CV for this exemplary assay according to the currentinvention over three measurements shows that the imprecision of theassay is determined only by counting error. This observation suggeststhat this assays according to the current invention, in some cases, mayhave imprecision <20% as long as at least 25 active beads are detected,equating to an enzyme concentration of 4.5 aM.

TABLE 1 Average # [SbG] single Average % Measurement Poisson (aM)complexes active % CV % CV 0 1 0.0016% 87%  122%  0.35 3 0.0086% 75% 55%  0.7 5 0.0099% 63%  46%  3.5 22 0.0413% 10%  21%  7 38 0.0713% 15% 16%  35 237 0.4461% 1% 7% 70 385 0.8183% 5% 5% 350 1787 3.3802% 2% 2%700 4036 7.5865% 5% 2% 3500 15634 30.6479% 3% 1% 7000 24836 44.5296% 1%1%

To quantify the potential sensitivity that may be achieved bysingulating enzyme-labeled molecules compared to conventional ensemblemeasurements, a model sandwich assay was developed to capture enzymemolecules on beads; the population of beads were either singulated andread using methods of the current invention, or read as an ensemblepopulation on a conventional plate reader. Beads were functionalizedwith DNA capture molecules, and subsequently saturated with biotinylatedcomplementary DNA target molecules in a one-step hybridization (seeMethods section below). These beads were used to capture variousconcentrations of an enzyme conjugate, streptavidin-β-galactosidase(SβG), commonly used as a label in ELISA. While the exemplary assayaccording to the current invention and conventional assay incubationswere performed under the substantially similar conditions, in theseexample the assays diverged at the bead readout step. For thecomparative conventional assay, beads were read out in 100 uL on afluorescence plate reader after 1 h incubation with 100 μMresorufin-β-D-galactopyranoside (RGP), a fluorogenic substrate forβ-galactosidase. The detection limit of the capture assay on themicrotiter plate reader was 15 fM of SβG (FIG. 24). For single moleculedetection according to the present invention, the beads were loaded intothe femtoliter arrays and, after sealing a solution of RGP into thewells of the array, signal generated from single enzymes accumulated inthe reaction chambers for 2.5 min, with fluorescent images acquiredevery 30 s. A white light image of the array was acquired at the end ofthe experiment. These images were analyzed to identify wells thatcontained beads (from the white light image) and determine which ofthose beads had an associated enzyme molecule bound (from time-lapsedfluorescent images). FIG. 24 shows a log-log plot of the percentage ofbeads that contained an enzyme as a function of bulk SβG concentration.The limit of detection (LOD) for the assay according to the inventionwas 220 zeptomolar (13 molecules in 100 μL, or 22 yoctomoles),corresponding to an improvement in sensitivity over the plate reader ofa factor of 68,000, showing that singulation can result in a dramaticincrease in sensitivity over conventional ensemble measurements. Theconcentration of SβG detected using the assay of the present inventionwas a factor of 140 lower than chemiluminescence detection of alkalinephosphatase (30 aM), the current most sensitive ELISA system. The highthermodynamic and kinetic efficiency that may be achieved for thepresent process (Table 2) can enable the detection of very low numbersof enzyme labels and indicates that the measurement of a single labeledmolecule from blood is a possibility.

TABLE 2 Calculations of the capture efficiency of enzyme label from 0.35aM to 7000 aM for the assay performed according to the invention (FIG.24). Column B Column D Column A Enzyme/bead Column C Background Column EAverage % active ratio from Total # of corrected # of Calculated # ofColumn F [SβG] beads observed Poisson enzymes on enzymes on enzymes inCapture (aM) (FIG. 2) distribution 400,000 beads beads 100-μL sampleEfficiency 0 0.0016% 0.000016 7 — — — 0.35 0.0086% 0.000086 34 28 21132%  0.7 0.0099% 0.000099 40 33 42 79% 3.5 0.0413% 0.000413 165 159 21175% 7 0.0713% 0.000713 285 279 421 66% 35 0.4461% 0.004471 1789 17822107 85% 70 0.8183% 0.008217 3287 3280 4214 78% 350 3.3802% 0.03438713755 13748 21070 65% 700 7.5865% 0.078897 31559 31552 42140 75% 350030.6479% 0.365974 146390 146383 210700 69% 7000 44.5296% 0.589320 235728235722 421400 56%In Table 2, for the experiments, on average 50,000 beads (-12.5%) wereinterrogated out of the 400,000 incubated with the solutions of enzyme.The low fraction of beads detected was limited by the number of reactionvessels used in this example (50,000 wells). By accounting for beadloss, the number of active beads observed experimentally (Column A) canbe used to estimate the total number of active beads out of the 400,000used (Column B). After background subtraction (Column C) and applying aPoisson distribution adjustment based on the distribution of 0, 1, 2, 3,4, etc., enzyme molecules per bead, the total number of moleculescaptured on beads can be determined (Column D). The ratio of this numberto the total number of enzymes in 100 uL at the start of the experiment(Column E; volume×concentration×Avogadro's number) yields a captureefficiency (Column F). The overall efficiency of capture and detectionof enzyme using the present assay is high (65-85%) and in someembodiments, may be minimally limited in this experiment by the slowdiffusion of the large SβG conjugate (MW˜515 kDa).

A sandwich-based assay was developed for two clinically-relevant proteinbiomarkers, namely prostate specific antigen (PSA) and tumor necrosisfactor-α (TNF-α). These assays show that the high enzyme labelsensitivity of the assay of this Example translates to highly sensitive(<1 fM) assays suitable for detecting proteins in blood. An assay forDNA was also developed to show that the assay of this Example can beused to directly detect single nucleic acid molecules without requiringreplication of the target. All assays were performed as outlined in FIG.23A and FIG. 23B, apart from the DNA assay where a capture sequence anda biotinylated signal sequence were used in place of capture anddetection antibodies, respectively. FIG. 25A, FIG. 25B and FIG. 25C showdata from the assays for A) PSA, B) TNF-α, and C) DNA. The human formsof the proteins were spiked into 25% bovine serum to be representativeof clinical test samples; a four-fold dilution factor was used which mayreduce matrix effects in immunoassays. DNA was detected in buffer to berepresentative of purified nucleic acid preparation techniques. Usingdigital detection to detect PSA in 25% serum, an LOD of 46 aM (1.5fg/mL) was attained, equating to 184 aM in whole serum. A leadingcommercial PSA assay (ADVIA Centaur, Siemens) reports an LOD of 3 pM(0.1 ng/mL) in human serum, and ultra-sensitive assays have beenreported with LODs in the range of 10-30 fM. The single molecule assayof the present Example was, therefore, more sensitive than thecommercial assay by a factor of 15,000, and more sensitive than otherultra-sensitive methods by a factor of at least 50. The detection limitin the TNF-α determination was 148 aM (2.5 fg/mL), corresponding to 590aM in whole serum. The highest sensitivity commercially-available ELISAfor TNF-α has an LOD of 21 fM (0.34 pg/mL) in serum (Table 1). The assayof the present Example, therefore, imparted an improvement over the mostsensitive TNF-α assay of a factor of 35. The LOD of the digital DNAsandwich assay was 135 aM, corresponding to about 8000 copies. Theability of certain assays of the present invention to potentiallymeasure much lower concentrations of proteins compared to conventionaltechniques arises from the extremely low background signals that may beachieved by digitizing the detection of proteins.

FIG. 25A, FIG. 25B and FIG. 25C show the attomolar detection of proteinsin serum and DNA in buffer using digital detection. Plots of % activebeads against analyte concentration for: (FIG. 25A) human PSA spikedinto 25% serum, (FIG. 25B) human TNF-α spiked into 25% serum, and (FIG.25C) DNA in buffer. The bottom row of plots shows the low-end of theconcentration range. Assays were performed by sequentially incubatingspecific capture beads with target solution, biotinylated detector, andSβG conjugate. After completion of the assay, beads were loaded intofemtoliter well arrays and interrogated for the presence of singleimmunocomplexes.

Background in the inventive digital detection immunoassays may arise, atleast in part from non-specific binding (NSB) of detection antibody andenzyme conjugate to the capture bead surface (Table 3). Because theinventive assays can provide improved label sensitivity overconventional assays (FIG. 24), significantly less detection antibody (˜1nM) and enzyme conjugate (1-50 pM) can be used to detect binding eventscompared to conventional assays (labeling reagent concentrations ˜10nM). The decreased label concentration may result in substantiallyreduced NSB to the capture surface, resulting in much lower backgroundsignals and lower LODs. For example, in the TNF-α and PSA determinationsas described above, the NSB levels were equivalent to the signalproduced by 1.8 fM and 1.2 fM of target protein, respectively. Thehighest sensitivity commercial TNF-α assay has an NSB level equivalentto 85 fM of TNF-α (FIG. 26), a factor of 50 higher. The ability toreduce backgrounds in certain assays of the present invention bylowering the concentration of labeling reagents can translate toimmunoassays with improved sensitivity over conventional assays.

TABLE 3 NSB dropout data. NSB Dropout Experiment (SiMoA PSA assay)Average SD CV % NSB No dAb; No PSA 0.110% 0.162% 147% from SbG 20% NoSbG; No PSA 0.000% 0.000% — from dAb 80% NSB 0.541% 0.194%  36% TotalNSB 100% In Table 3, a dropout experiment isolating the sources of NSB in the PSAdigital assay. By comparing ‘no detection antibody NSB’ (No dAb) and ‘noSβG NSB’ (No SβG) to total NSB (NSB), the contribution of detectionantibody and SβG to the NSB of the assay was determined.

To demonstrate the unique diagnostic measurements that could be affordedby detecting single molecules of a protein in human clinical samples,PSA was measured in serum samples from patients who had undergoneradical prostatectomy (RP). PSA is a serum biomarker for prostate cancerused as both a screening tool and to monitor for the biochemicalrecurrence of patients who have undergone radical prostatectomy. Afterradical prostatectomy, the vast majority of PSA is eliminated, andlevels fall below the detection limit of standard commercial assays (3pM or 0.1 ng/mL). Regular monitoring of these patients for return of PSAcan detect recurrence of prostate cancer, but several years may passpost-surgery for biochemical recurrence to be detected by currentimmunoanalyzers. The ability to accurately quantify PSA levels inpost-prostatectomy patients at very low concentrations (<3 fM or 100fg/mL) may provide early indication of recurrence should PSA levelsincrease. FIG. 27 shows PSA levels measured using the assay of thisExample in the serum of thirty patients (age 60-89) who had undergoneradical prostatectomy and whose blood was collected at least 6 weekspost-surgery. The PSA levels in the sera of all 30 patients were belowthe detection limit of commercial assays. Here, whole serum samples werediluted 1:4 in buffer and measured using the PSA digital assay of thisExample (FIG. 25A). PSA was successfully detected in all 30 patientsusing the present assay. Nine of the thirty samples fell below the LODof the previously highest sensitivity PSA assay. These data demonstratea potential clinical utility of certain inventive assays for measuringproteins in serum at concentrations well below the capability of currenttechnology. Table 4 summarizes the patient results.

FIG. 27 shows digital detection of PSA in serum samples of patients whohad undergone radical prostatectomy. The concentrations of PSA weredetermined using the assay of the present Example in serum samples fromRP patients (circles (iv)), healthy control samples (circles (ii)), andBio-Rad PSA control samples (circles (i)). RP patient samples wereobtained from SeraCare Life Sciences (Milford, Mass.) and all hadundetectable PSA levels as measured by a leading clinical diagnosticassay (ADVIA Centaur); line (iv) represents the detection limit of theADVIA Centaur PSA assay (100 pg/mL or 3 pM). All 30 patient samples wereabove the detection limit of the inventive PSA digital assay, shown bythe line (v) (0.00584 pg/mL or 184 aM), with the lowest patient PSAconcentrations measured at 0.014 pg/mL (420 aM) using the present assay.Patient samples with the lowest PSA levels were detectable, butapproached the LOD of the assay resulting in a high dose % CV. Thepresent assay was validated for specificity to PSA using controlstandards (Bio-Rad) and serum from healthy individuals (ProMedDx) thathad been assayed using the ADVIA Centaur PSA assay (See Table 5).

TABLE 4 Summary of Patient Results Patient [PSA] Dose ID (pg/mL) % CVS600 9.39  6% S599 0.75 10% S598 2.71 12% S597 1.79 12% S596 2.46 17%S595 0.32 21% S594 1.63 15% S593 1.15 12% S592 3.46  9% S591 0.21 25%S590 0.22 22% S589 0.85 17% S588 2.33  3% S587 1.06 13% S586 1.29 22%S585 0.49 84% S584 0.056 136%  S583 1.33 26% S582 4.76  9% S581 1.57 31%S580 0.30  4% S579 1.22 15% S578 0.090 91% S577 1.92  6% S576 0.014286%  S575 0.79 63% S574 1.62 20% S573 0.22 32% S572 1.04 20% S571 0.2421%

TABLE 5 Assay Method of the Centaur (ng/mL) present invention (ng/mL)Bio-Rad Control 1 0.838 1.06 ± 0.21 Bio-Rad Control 2 2.47 2.66 ± 0.36Normals ProMedDx S376 2.1 1.60 ProMedDx S378 2.3 1.70 ProMedDx S381 2.92.14 ProMedDx S388 4.1 3.95 ProMedDx S395 0.93 0.63 ProMedDx S396 0.90.77 ProMedDx S397 1.2 0.66Table 5 shows the specificity of the present assay was confirmed usingPSA samples from Bio-Rad (controls) and ProMedDx (serum from healthyindividuals) that had previously been tested on a commercialimmunoanalyzer (ADVIA Centaur, Siemens). The PSA concentrations of thehealthy serum samples determined using the exemplary method were(24±12)% lower than those originally determined on the ADVIA Centaur.There are two possible explanations for this systematic bias between thetwo technologies. First, the ADVIA Centaur values were obtained on freshserum, whereas values for the present assay were obtained after the serahad been frozen for extended periods of time and had experienced afreeze-thaw cycle. Second, there may be differences between the PSAcalibrators used to generate the calibration curves that would result indifferences in PSA concentrations determined. Complex PSA from the WorldHealth Organization (WHO) was used as calibrators; the ADVIA Centaurcalibration PSA is unknown.

By isolating and detecting single immunocomplexes formed in ELISA,certain assays of the invention can impart sensitivity, precision, anddynamic range improvements over standard readout methods and other knownultra-sensitive approaches. While certain of the inventive assays canprovide sensitivity below the detectable limit of standard andultra-sensitive readout formats in serum, potentially two more logs ofsensitivity may be available based on the enzyme label LOD (FIG. 24).The ability to isolate and interrogate single molecules on individualbeads according to certain embodiments of the invention may facilitatedistinguishing true antibody-antigen binding events fromnon-specifically bound complexes. Identifying and differentiating thesetwo populations may permit the detection of a single biomarker moleculein a human serum sample.

Capture of proteins on magnetic beads and formation of enzyme-labeledimmunocomplex (FIGS. 25 and 27). Beads functionalized with an antibodyto the target protein were prepared according to the manufacturer'sinstructions. Test solutions containing the protein of interest wereincubated with suspensions of 200,000 magnetic beads for 2 h at roomtemperature. The beads were then separated and washed three times in PBSand 0.1% Tween-20. The beads were resuspended and incubated withsolutions containing detection antibody (typically, 1-3 nM) for 45 minat room temperature. The beads were then separated and washed threetimes in PBS and 0.1% Tween-20. The beads were incubated with solutionscontaining SβG (1-50 pM) for 30 min at room temperature, separated, andwashed six times in PBS and 0.1% Tween-20. The beads were thenresuspended in 10 μL of PBS and loaded onto a femtoliter well array.

EXAMPLE 20

FIG. 28 shows a histogram of the average fluorescence intensity ofreaction vessels in an experiment of the present invention. Arepresentative set of images from the experiment were analyzed todetermine populations of reaction vessels that: (i) contained no beads;(ii) contained a bead (from white light scatter) but no enzyme (noincrease in fluorescence intensity); and, (iii) contained a bead (fromwhite light scatter) and an enzyme (increasing fluorescence intensityover four consecutive images, and an overall increase in fluorescence ofat least 20%). Specifically, line (i) represents data from emptyreaction vessels, line (ii) represents reaction vessels with a bead butno enzyme (“off” beads), and line (iii) represents reaction vessels withboth a bead and enzymatic activity (“on” beads).

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively.

1. A method for determining a measure of the concentration of analytemolecules or particles in a fluid sample, comprising: exposing aplurality of capture objects that each include a binding surface havingaffinity for at least one type of analyte molecule or particle, to asolution containing or suspected of containing the at least one type ofanalyte molecules or particles; immobilizing analyte molecules orparticles with respect to the plurality of capture objects such that atleast some of the capture objects associate with at least one analytemolecule or particle and a statistically significant fraction of thecapture objects do not associate with any analyte molecule or particle;spatially segregating at least a portion of the capture objectssubjected to the immobilizing step into a plurality of separatelocations; addressing at least a portion of the plurality of locationssubjected to the spatially segregating step and determining the numberof said locations containing an analyte molecule or particle; anddetermining a measure of the concentration of analyte molecules orparticles in the fluid sample based at least in part on the number oflocations determined to contain an analyte molecule or particle.
 2. Amethod for determining a measure of the concentration of analytemolecules or particles in a fluid sample, comprising: exposing aplurality of capture objects that each include a binding surface havingaffinity for at least one type of analyte molecule or particle, to asolution containing or suspected of containing the at least one type ofanalyte molecules or particles to form capture objects comprising atleast one immobilized analyte molecule or particle; mixing the captureobjects prepared in the exposing step to a plurality of binding ligandssuch that at least some of the capture objects associate with a singlebinding ligand and a statistically significant fraction of the captureobjects do not associate with any binding ligand; spatially segregatingat least a portion of the capture objects subjected to the mixing stepinto a plurality of locations; addressing at least a portion of theplurality of locations subjected to the spatially segregating step anddetermining the number of locations containing a binding ligand; anddetermining a measure of the concentration of analyte molecules orparticles in the fluid sample based at least in part on the number oflocations determined to contain a binding ligand.
 3. The method of claim1, wherein the percentage of capture objects which associate with atleast one analyte molecule and/or binding ligand is less than about 50%,less than about 40%, less than about 30%, less than about 20%, less thanabout 10%, or less than about 5%, or less than about 1%, or less thanabout 0.5%, or less than about 0.1% or the total number of captureobjects.
 4. The method of claim 1, wherein the percentage of captureobjects which do not associated with any analyte molecules and/orbinding ligands is at least about 20%, or at least about 40%, or atleast about 50%, or at least about 60%, or at least about 70%, or atleast about 75%, or at least about 80%, or at least about 90%, or atleast about 95%, or at least about 99%, or at least about 99.5%, or atleast about 99.9%, of the total number of capture objects.
 5. The methodof claim 1, wherein in the addressing step, the number of said locationscontaining a capture object that includes a binding surface havingaffinity for at least one type of analyte molecule or particle notcontaining an analyte molecule or particle or a binding ligand isdetermined.
 6. The method of claim 5, wherein the measure of theconcentration of analyte molecule or particles in the fluid sample isbased at least in part on the ratio of the number of locations addressedin the addressing step determined to contain a capture object thatincludes a binding surface having affinity for at least one type ofanalyte molecule or particle containing an analyte molecule or particleor a binding ligand, to the total number of locations addressed in theaddressing step determined to contain a capture object that includes abinding surface having affinity for at least one type of analytemolecule or particle.
 7. The method of claim 5, wherein the measure ofthe concentration of analyte molecule or particles in the fluid sampleis based at least in part on the ratio of the number of locationsaddressed in the addressing step determined to contain a capture objectthat includes a binding surface having affinity for at least one type ofanalyte molecule or particle containing an analyte molecule or particleor a binding ligand, to the number of locations addressed in theaddressing step determined to contain a capture object that includes abinding surface having affinity for at least one type of analytemolecule or particle but not to contain any capture objects that includea binding surface having affinity for at least one type of analytemolecule or particle containing an analyte molecule or particle or abinding ligand.
 8. The method of claim 1, wherein the measure of theconcentration of analyte molecules or particles in the fluid sample isbased at least in part on the ratio of the number of locations addressedin the addressing step determined to contain a capture object thatincludes a binding surface having affinity for at least one type ofanalyte molecule or particle containing an analyte molecule or particleor binding ligand, to the number of locations addressed in theaddressing step that do not contain a capture object that includes abinding surface having affinity for at least one type of analytemolecule or particle.
 9. The method of claim 1, wherein the plurality ofcapture objects that include a binding surface having affinity for atleast one type of analyte molecule or particle comprises a plurality ofbeads.
 10. The method of claim 1, wherein the plurality of locationscomprises a plurality of reaction vessels.
 11. The method of claim 10,wherein the plurality of reaction vessels is formed on the end of afiber optic bundle.
 12. The method of claim 1, wherein at least aportion of the analyte molecules or particles are associated with atleast one binding ligand.
 13. The method of claim 1, wherein the analytemolecules or particles comprise an enzymatic component.
 14. The methodof claim 2, wherein the binding ligand comprises an enzymatic component.15. The method of claim 1, wherein the concentration of analytemolecules or particles in the fluid sample is less than about 50×10⁻¹⁵M, or less than about 40×10⁻¹⁵ M, or less than about 30×10⁻¹⁵ M, or lessthan about 20×10⁻¹⁵ M, or less than about 10×10⁻¹⁵ M, or less than about5×10⁻¹⁵ M, or less than about 1×10⁻¹⁵ M.
 16. The method of claim 1,wherein the measure of the concentration of analyte molecules orparticles in the fluid sample is determined at least in part bycomparison of a measured parameter to a calibration standard.
 17. Themethod of claim 1, wherein during the immobilizing step, at least about10%, or at least about 20%, or at least about 30%, or at least about40%, or at least about 50%, or at least about 60%, or at least about70%, or at least about 80%, or at least about 90%, or at least about 95%of the analyte molecules or particles are immobilized with respect to acapture object that includes a binding surface having affinity for atleast one type of analyte molecule or particle.
 18. The method of claim2, wherein during the mixing step, at least about 10%, or at least about20%, or at least about 30%, or at least about 40%, or at least about50%, or at least about 60%, or at least about 70%, or at least about80%, or at least about 90% of the immobilized analyte molecules orparticles associate with a binding ligand.
 19. The method of claim 1,wherein during the spatially segregating step, at least about 5%, or atleast about 10%, or at least about 20%, or at least about 30%, or atleast about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% of thecapture objects subjected to the immobilizing steps are spatiallyseparated into the plurality of locations.
 20. The method of claim 10,wherein the number of reaction vessels addressed in the addressing stepis at least about 5%, or at least about 10%, or at least about 20%, orat least about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90% of the total number of reaction vessels. 21-141.(canceled)